token_classification/10-1038_emboj-2010-237.xml

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<article doi="10.1038/emboj.2010.237" abstract="The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders."><fig id="1931"><title></title><label>Figure 1</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1931"/><sd-panel panel_id="5034"><b>(A)</b> <span><sd-tag id="sdTag547" source="sdapi" category="entity" entity_type="protein" role="assayed" text="EGFR" ext_ids="Q01279" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Egfr" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">EGFR</sd-tag> degradation was followed in</span> <sd-tag id="sdTag557" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="MSD" ext_ids="10090///GO:0036020" norm_text="" ext_dbs="NCBI taxon///Gene Ontology" in_caption="True" ext_names="Mus musculus///endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy////http://amigo.geneontology.org/amigo/term/">MSD</sd-tag><span>,</span> <sd-tag id="sdTag42">MPS‐IIIA</sd-tag> <span>and WT</span> <sd-tag id="sdTag6" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> <span>by treating the cells with</span> <sd-tag id="sdTag7" source="sdapi" category="entity" entity_type="protein" role="intervention" text="EGF" ext_ids="P01132" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Egf" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">EGF</sd-tag> <span>for the indicated time to stimulate</span> <span><sd-tag id="sdTag549" source="sdapi" category="entity" entity_type="protein" role="assayed" text="EGFR" ext_ids="Q01279" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Egfr" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">EGFR</sd-tag></span> <span>internalization. The cells were then immediately lysed and subjected to anti‐<sd-tag id="sdTag548" source="sdapi" category="entity" entity_type="protein" role="assayed" text="EGFR" ext_ids="Q01279" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Egfr" ext_tax_ids="" ext_tax_names="" ext_urls="https://www.uniprot.org/uniprot/">EGFR</sd-tag> blotting. The amount of remaining</span> <span><sd-tag id="sdTag550" source="sdapi" category="entity" entity_type="protein" role="assayed" text="EGFR" ext_ids="Q01279" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Egfr" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">EGFR</sd-tag></span> <span>was quantified by</span> <sd-tag id="sdTag8">densitometry analysis</sd-tag> <span>(Image J) of the</span> <sd-tag id="sdTag9" source="sdapi" category="assay" entity_type="" role="" text="blot" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">blot</sd-tag> <span>and expressed in the chart as % of the</span> <span><sd-tag id="sdTag551" source="sdapi" category="entity" entity_type="protein" role="assayed" text="EGFR" ext_ids="Q01279" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Egfr" ext_tax_ids="" ext_tax_names="" ext_urls="https://www.uniprot.org/uniprot/">EGFR</sd-tag></span> <span>amount present at time T</span><sub>0</sub> <span>(100%). The values in the chart represent the mean±s.e.m. values of three independent experiments.</span><graphic href="https://api.sourcedata.io/file.php?panel_id=5034"/></sd-panel><sd-panel panel_id="5035"><b>(B)</b> <sd-tag id="sdTag16" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, <sd-tag id="sdTag17" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> and WT <sd-tag id="sdTag18" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> cells loaded with <sd-tag id="sdTag13" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="dextran" ext_ids="CHEBI:52071" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="dextran" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">dextran</sd-tag> (<sd-tag id="sdTag14" source="sdapi" category="assay" entity_type="" role="" text="alexafluor‐594" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">alexafluor‐594</sd-tag> conjugated) were labelled with anti‐<sd-tag id="sdTag15" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LAMP1" ext_ids="P11438" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Lamp1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">LAMP1</sd-tag> antibody and the percentage of <sd-tag id="sdTag21" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="dextran" ext_ids="CHEBI:52071" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="dextran" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">dextran</sd-tag> co‐localizing with <sd-tag id="sdTag22" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LAMP1" ext_ids="P11438" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Lamp1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">LAMP1</sd-tag> was evaluated. The chart displays merge values (means±s.e.m.) that represent the percentage of <sd-tag id="sdTag552" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="dextran" ext_ids="CHEBI:52071" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="dextran" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">dextran</sd-tag> co‐localizing with <sd-tag id="sdTag553" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LAMP1" ext_ids="P11438" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Lamp1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">LAMP1</sd-tag> measured in 15 different cells of triplicate experiments.<graphic href="https://api.sourcedata.io/file.php?panel_id=5035"/></sd-panel><sd-panel panel_id="5036"><b>(C)</b> The rate of <sd-tag id="sdTag24" source="sdapi" category="entity" entity_type="subcellular" role="component" text="lysosome" ext_ids="GO:0005764" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="lysosome" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">lysosome</sd-tag> fusion with <sd-tag id="sdTag25" source="sdapi" category="entity" entity_type="subcellular" role="component" text="autophagosomes" ext_ids="GO:0032009" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="early phagosome" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">autophagosomes</sd-tag> was monitored in <sd-tag id="sdTag29" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, <sd-tag id="sdTag30" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> and WT <sd-tag id="sdTag31" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> transfected with a tandem fluorescently tagged <sd-tag id="sdTag26" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LC3" ext_ids="Q9CQV6///Q91VR7" norm_text="" ext_dbs="Uniprot///Uniprot" in_caption="True" ext_names="Map1lc3b///Map1lc3a" ext_tax_ids="10090///10090" ext_tax_names="Mus musculus///Mus musculus" ext_urls="https://www.uniprot.org/uniprot////https://www.uniprot.org/uniprot/">LC3</sd-tag> ( Kimura <i>et al</i>, 2007). The rate of <sd-tag id="sdTag27" source="sdapi" category="entity" entity_type="subcellular" role="component" text="autophagosome" ext_ids="GO:0032009" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="early phagosome" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">autophagosome</sd-tag> maturation reflected the percentage of the <sd-tag id="sdTag33" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LC3" ext_ids="Q9CQV6///Q91VR7" norm_text="" ext_dbs="Uniprot///Uniprot" in_caption="True" ext_names="Map1lc3b///Map1lc3a" ext_tax_ids="10090///10090" ext_tax_names="Mus musculus///Mus musculus" ext_urls="https://www.uniprot.org/uniprot////https://www.uniprot.org/uniprot/">LC3</sd-tag> 'unfused' (green/red fluorescence ratio) at each time (<sd-tag id="sdTag38" source="sdapi" category="time" entity_type="" role="" text="1 and 3 h" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">1 and 3 h</sd-tag>) after <sd-tag id="sdTag28" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="bafilomycin" ext_ids="CHEBI:22689" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="bafilomycin A1" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">bafilomycin</sd-tag> removal (<i>T</i><sub>0</sub>). The percentage of <sd-tag id="sdTag554" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LC3" ext_ids="Q9CQV6///Q91VR7" norm_text="" ext_dbs="Uniprot///Uniprot" in_caption="True" ext_names="Map1lc3b///Map1lc3a" ext_tax_ids="10090///10090" ext_tax_names="Mus musculus///Mus musculus" ext_urls="https://www.uniprot.org/uniprot////https://www.uniprot.org/uniprot/">LC3</sd-tag> 'unfused' was displayed versus the value at <i>T</i><sub>0</sub> (assumed to be 100%). Values are represented as means±s.e.m. of triplicate experiments. <sup>*</sup><i>P</i>0.05, Student's <i>t</i>‐test: (<b>A</b>): WT versus <sd-tag id="sdTag36" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and WT versus <sd-tag id="sdTag37" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag>; (<b>B</b>, <b>C</b>): WT versus <sd-tag id="sdTag555" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and WT versus <sd-tag id="sdTag556" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> at each time point. Scale bar: 10 μm (<b>B</b>, <b>C</b>).<graphic href="https://api.sourcedata.io/file.php?panel_id=5036"/></sd-panel></fig><fig id="1932"><title></title><label>Figure 2</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1932"/><sd-panel panel_id="5037"><b>(A)</b> <sd-tag id="sdTag40" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="Cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">Cholesterol</sd-tag> levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of <sd-tag id="sdTag558" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> per μg of protein (see Materials and methods section for details).<graphic href="https://api.sourcedata.io/file.php?panel_id=5037"/></sd-panel><sd-panel panel_id="5038"><b>(B)</b> <span><sd-tag id="sdTag534">Filipin staining</sd-tag> showing</span> <sd-tag id="sdTag61" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> accumulation in the <sd-tag id="sdTag54" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="endolysosomal" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">endolysosomal</sd-tag> compartment of <sd-tag id="sdTag55" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and <sd-tag id="sdTag56" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> <sd-tag id="sdTag57" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> (arrowheads and enlarged images).<graphic href="https://api.sourcedata.io/file.php?panel_id=5038"/></sd-panel><sd-panel panel_id="5039"><b>(C, D)</b> Total lipids extracted from the indicated <sd-tag id="sdTag62" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="endolysosomal membrane" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">endolysosomal membrane</sd-tag> samples (30 μg of proteins) were either (<b>C</b>) subjected to a <sd-tag id="sdTag63" source="sdapi" category="assay" entity_type="" role="" text="phosphate assay" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">phosphate assay</sd-tag> to quantify the bulk of <sd-tag id="sdTag64" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="phospholipids" ext_ids="CHEBI:16247" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="phospholipid" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">phospholipids</sd-tag> or (<b>D</b>) separated by <sd-tag id="sdTag65" source="sdapi" category="assay" entity_type="" role="" text="TLC" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">TLC</sd-tag>. Phospholipids and <sd-tag id="sdTag79" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> on <sd-tag id="sdTag78" source="sdapi" category="assay" entity_type="" role="" text="TLC" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">TLC</sd-tag> plates were revealed by <sd-tag id="sdTag66" source="sdapi" category="assay" entity_type="" role="" text="molybdenum blue staining" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">molybdenum blue staining</sd-tag>. CHOL, <sd-tag id="sdTag559" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag>; LBPA <sd-tag id="sdTag82" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="lysobisphosphatidic acid" ext_ids="CHEBI:60815" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="lysobisphosphatidic acid" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">lysobisphosphatidic acid</sd-tag>; PC, <sd-tag id="sdTag83" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="phosphatidylcoline" ext_ids="CHEBI:64482" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="phosphatidylcholine" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">phosphatidylcoline</sd-tag>; PE <sd-tag id="sdTag84" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="phosphatidylethanolamine" ext_ids="CHEBI:16038" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="phosphatidylethanolamine" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">phosphatidylethanolamine</sd-tag>; PI <sd-tag id="sdTag85" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="phosphatidyinositol" ext_ids="CHEBI:28874" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="phosphatidylinositol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">phosphatidyinositol</sd-tag>; SM <sd-tag id="sdTag86" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="sphingomyelin" ext_ids="CHEBI:64583" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="sphingomyelin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">sphingomyelin</sd-tag>. (<b>A</b>, <b>C</b>) Values represent the mean±s.e.m. values of three independent experiments. <sup>*</sup><i>P</i>0.05, Student's <i>t</i>‐test: (<b>A</b>, <b>C</b>): WT versus <sd-tag id="sdTag80" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and WT versus <sd-tag id="sdTag81" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag>. Scale bar, 10 μm (<b>B</b>).<graphic href="https://api.sourcedata.io/file.php?panel_id=5039"/></sd-panel></fig><fig id="1933"><title></title><label>Figure 3</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1933"/><sd-panel panel_id="8224"><sd-tag id="sdTag88" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="Cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">Cholesterol</sd-tag> accumulation inhibits lysosomal fusion. <sd-tag id="sdTag87" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="Endolysosomal membrane" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">Endolysosomal membrane</sd-tag> <sd-tag id="sdTag89" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> <span>measurements and <sd-tag id="sdTag581" source="sdapi" category="assay" entity_type="" role="" text="Filipin staining" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Filipin staining</sd-tag> were carried out in either (</span><strong>A</strong>) WT <sd-tag id="sdTag91" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> loaded with <sd-tag id="sdTag92" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> or in <sd-tag id="sdTag93">MSD</sd-tag><graphic href="https://api.sourcedata.io/file.php?panel_id=8224"/></sd-panel><sd-panel panel_id="8230"><sd-tag id="sdTag412" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">Cholesterol</sd-tag> accumulation inhibits lysosomal fusion. <sd-tag id="sdTag413" source="sdapi" category="entity" entity_type="subcellular" role="component" text="Endolysosomal membrane" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">Endolysosomal membrane</sd-tag> <sd-tag id="sdTag414" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> measurements and <sd-pretag id="sdPretag14588126706520" parent-tag-id="533">Filipin staining</sd-pretag> were carried out in either (<strong>A</strong>) WT <sd-tag id="sdTag416" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> loaded with <sd-tag id="sdTag417" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> or in <sd-pretag id="sdPretag14728261260400" parent-tag-id="542">MSD</sd-pretag> and (<strong>D</strong>) <sd-pretag id="sdPretag14313355644610" parent-tag-id="507">MPS‐IIIA</sd-pretag> <sd-tag id="sdTag420" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> treated with <sd-pretag id="sdPretag14313354906020" parent-tag-id="505">MβCD</sd-pretag>. Arrowheads and enlarged images show <sd-tag id="sdTag423" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> accumulation in endolysosomes of <sd-tag id="sdTag574" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag>‐loaded WT <sd-tag id="sdTag424" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>. After treatments the rate of <sd-tag id="sdTag427" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="autophagosome" ext_ids="GO:0032009" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="early phagosome" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">autophagosome</sd-tag> maturation (<strong>B</strong>, <strong>E</strong>) and the transport of fluorescent dextran to lysosomes (<strong>C</strong>, <strong>F</strong>) were also analysed as in Figure 1. WT controls for <sd-tag id="sdTag575" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="autophagosome" ext_ids="GO:0032009" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="early phagosome" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">autophagosome</sd-tag> maturation and dextran transport experiments were performed as shown in Figure 1. (<strong>A</strong>-<strong>F</strong>) Values represent the mean±s.e.m. values of three independent experiments. <sup>*</sup><em>P</em>0.05, Student's <em>t</em>‐test: (<strong>A</strong>, <strong>C</strong>): WT versus WT+<sd-tag id="sdTag430" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag>; (<strong>B</strong>): WT versus WT+<sd-tag id="sdTag576" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> for each time point; (<strong>D</strong>, <strong>F</strong>): <sd-pretag id="sdPretag14728260544030" parent-tag-id="542">MSD</sd-pretag> versus <sd-pretag id="sdPretag14728260544031" parent-tag-id="542">MSD</sd-pretag>+<sd-pretag id="sdPretag14313355552330" parent-tag-id="505">MβCD</sd-pretag> and <sd-pretag id="sdPretag14313354900850" parent-tag-id="507">MPS‐IIIA</sd-pretag> versus <sd-pretag id="sdPretag14313354900851" parent-tag-id="507">MPS‐IIIA</sd-pretag>+<sd-pretag id="sdPretag14313355552331" parent-tag-id="505">MβCD</sd-pretag>; (<strong>E</strong>): <sd-pretag id="sdPretag14728260406660" parent-tag-id="542">MSD</sd-pretag> versus <sd-pretag id="sdPretag14728260406661" parent-tag-id="542">MSD</sd-pretag>+<sd-pretag id="sdPretag14313355418540" parent-tag-id="505">MβCD</sd-pretag> and <sd-pretag id="sdPretag14313355381420" parent-tag-id="507">MPS‐IIIA</sd-pretag> versus <sd-pretag id="sdPretag14313355381421" parent-tag-id="507">MPS‐IIIA</sd-pretag>+<sd-pretag id="sdPretag14313355418541" parent-tag-id="505">MβCD</sd-pretag> for each time point. Scale bar<graphic href="https://api.sourcedata.io/file.php?panel_id=8230"/></sd-panel><sd-panel panel_id="8231"><p><sd-tag id="sdTag434" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">Cholesterol</sd-tag> accumulation inhibits lysosomal fusion. Endolysosomal membrane <sd-tag id="sdTag438" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> measurements and <sd-tag id="sdTag535" source="sdapi" category="assay" entity_type="" role="" text="Filipin staining" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Filipin staining</sd-tag> were carried out in either (<strong>A</strong>) WT <sd-tag id="sdTag442" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> loaded with <sd-tag id="sdTag444" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> or in <sd-tag id="sdTag446" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and (<strong>D</strong>) <sd-tag id="sdTag448" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> <sd-tag id="sdTag450" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> treated with <sd-tag id="sdTag452" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag>. Arrowheads and enlarged images show <sd-tag id="sdTag456" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> accumulation in endolysosomes of <sd-tag id="sdTag577" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag>‐loaded WT <sd-tag id="sdTag458" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>. After treatments the rate of <sd-tag id="sdTag460" source="sdapi" category="entity" entity_type="subcellular" role="component" text="autophagosome" ext_ids="GO:0032009" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="early phagosome" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">autophagosome</sd-tag> maturation (<strong>B</strong>, <strong>E</strong>) and the transport of fluorescent <sd-tag id="sdTag462" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="dextran" ext_ids="CHEBI:52071" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="dextran" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">dextran</sd-tag> to lysosomes (<strong>C</strong>, <strong>F</strong>) were also analysed as in Figure 1. WT controls for <sd-tag id="sdTag464" source="sdapi" category="entity" entity_type="subcellular" role="component" text="autophagosome" ext_ids="GO:0032009" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="early phagosome" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">autophagosome</sd-tag> maturation and <sd-tag id="sdTag466" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="dextran" ext_ids="CHEBI:52071" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="dextran" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">dextran</sd-tag> transport experiments were performed as shown in Figure 1. (<strong>A</strong>-<strong>F</strong>) Values represent the mean±s.e.m. values of three independent experiments. <sup>*</sup><em>P</em>0.05, Student's <em>t</em>‐test: (<strong>A</strong>, <strong>C</strong>): WT versus WT+<sd-tag id="sdTag468" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag>; (<strong>B</strong>): WT versus WT+<sd-tag id="sdTag470" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> for each time point; (<strong>D</strong>, <strong>F</strong>): <sd-tag id="sdTag472" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> versus <sd-tag id="sdTag474" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+<sd-tag id="sdTag476" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> and <sd-tag id="sdTag480" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> versus <sd-tag id="sdTag578" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag>+MβCD; (<strong>E</strong>): MSD versus <sd-tag id="sdTag486" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+MβCD and <sd-tag id="sdTag490" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> versus MPS‐IIIA+<sd-tag id="sdTag494" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> for each time point. Scale bar: 10 μm (<strong>A</strong>, <strong>C</strong>, <strong>D</strong>, <strong>F</strong>).</p><graphic href="https://api.sourcedata.io/file.php?panel_id=8231"/></sd-panel><sd-panel panel_id="8232"><p><sd-pretag parent-tag-id="40" id="sdPretag14313348695970" class="component molecule">Cholesterol</sd-pretag> accumulation inhibits lysosomal fusion. <sd-pretag parent-tag-id="87" id="sdPretag14313348484700" class="component subcellular">Endolysosomal membrane</sd-pretag> <sd-pretag parent-tag-id="60" id="sdPretag14313348150470" class="component molecule">cholesterol</sd-pretag> measurements and <sd-pretag id="sdPretag14588126048910" parent-tag-id="533">Filipin staining</sd-pretag> were carried out in either (<strong>A</strong>) WT <sd-pretag parent-tag-id="6" id="sdPretag14313348868910" class="component cell">MEFs</sd-pretag> loaded with <sd-pretag parent-tag-id="60" id="sdPretag14313348773390" class="component molecule">cholesterol</sd-pretag> or in <sd-pretag parent-tag-id="11" id="sdPretag14313348274830" class="component organism">MSD</sd-pretag> and (<strong>D</strong>) <sd-pretag parent-tag-id="12" id="sdPretag14313348604310" class="component organism">MPS‐IIIA</sd-pretag> <sd-pretag parent-tag-id="6" id="sdPretag14313348867820" class="component cell">MEFs</sd-pretag> treated with <sd-pretag parent-tag-id="173" id="sdPretag14313348344550" class="component molecule">MβCD</sd-pretag>. Arrowheads and enlarged images show <sd-pretag parent-tag-id="60" id="sdPretag14313348733820" class="component molecule">cholesterol</sd-pretag> accumulation in endolysosomes of <sd-pretag parent-tag-id="60" id="sdPretag14313348733821" class="component molecule">cholesterol</sd-pretag>‐loaded WT <sd-pretag parent-tag-id="6" id="sdPretag14313348867821" class="component cell">MEFs</sd-pretag>. After treatments the rate of <sd-pretag parent-tag-id="27" id="sdPretag14313348805400" class="component subcellular">autophagosome</sd-pretag> maturation (<strong>B</strong>, <strong>E</strong>) and the transport of fluorescent <sd-pretag parent-tag-id="13" id="sdPretag14313348212810" class="component molecule">dextran</sd-pretag> to lysosomes (<strong>C</strong>, <strong>F</strong>) were also analysed as in Figure 1. WT controls for <sd-pretag parent-tag-id="27" id="sdPretag14313348744650" class="component subcellular">autophagosome</sd-pretag> maturation and <sd-pretag parent-tag-id="13" id="sdPretag14313349026050" class="component molecule">dextran</sd-pretag> transport experiments were performed as shown in Figure 1. (<strong>A</strong>-<strong>F</strong>) Values represent the mean±s.e.m. values of three independent experiments. <sup>*</sup><em>P</em>0.05, Student's <em>t</em>‐test: (<strong>A</strong>, <strong>C</strong>): WT versus WT+<sd-pretag parent-tag-id="60" id="sdPretag14313348529480" class="component molecule">cholesterol</sd-pretag>; (<strong>B</strong>): WT versus WT+<sd-pretag parent-tag-id="60" id="sdPretag14313348440700" class="component molecule">cholesterol</sd-pretag> for each time point; (<strong>D</strong>, <strong>F</strong>): <sd-pretag parent-tag-id="11" id="sdPretag14313348442730" class="component organism">MSD</sd-pretag> versus <sd-pretag parent-tag-id="11" id="sdPretag14313348442731" class="component organism">MSD</sd-pretag>+<sd-pretag parent-tag-id="173" id="sdPretag14313348316020" class="component molecule">MβCD</sd-pretag> and <sd-pretag parent-tag-id="12" id="sdPretag14313348612620" class="component organism">MPS‐IIIA</sd-pretag> versus <sd-pretag parent-tag-id="12" id="sdPretag14313348612621" class="component organism">MPS‐IIIA</sd-pretag>+<sd-pretag parent-tag-id="173" id="sdPretag14313349085700" class="component molecule">MβCD</sd-pretag>; (<strong>E</strong>): <sd-pretag parent-tag-id="11" id="sdPretag14313348380560" class="component organism">MSD</sd-pretag> versus <sd-tag id="sdTag506" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+<sd-tag id="sdTag505" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> and <sd-tag id="sdTag507" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> versus <sd-pretag parent-tag-id="12" id="sdPretag14313348612381" class="component organism">MPS‐IIIA</sd-pretag>+<sd-pretag parent-tag-id="173" id="sdPretag14313348741420" class="component molecule">MβCD</sd-pretag> for each time point. Scale bar: 10 μm (<strong>A</strong>, <strong>C</strong>, <strong>D</strong>, <strong>F</strong>).</p><graphic href="https://api.sourcedata.io/file.php?panel_id=8232"/></sd-panel><sd-panel panel_id="8233"><sd-pretag id="sdPretag14313349131190" parent-tag-id="40" class="component molecule">Cholesterol</sd-pretag> accumulation inhibits lysosomal fusion. <sd-pretag id="sdPretag14313349473210" parent-tag-id="87" class="component subcellular">Endolysosomal membrane</sd-pretag> <sd-pretag id="sdPretag14313349259170" parent-tag-id="60" class="component molecule">cholesterol</sd-pretag> measurements and <sd-pretag id="sdPretag14588126802180" parent-tag-id="533">Filipin staining</sd-pretag> were carried out in either (<strong>A</strong>) WT <sd-pretag id="sdPretag14313349479700" parent-tag-id="6" class="component cell">MEFs</sd-pretag> loaded with <sd-pretag id="sdPretag14313349252610" parent-tag-id="60" class="component molecule">cholesterol</sd-pretag> or in <sd-pretag id="sdPretag14313348606820" parent-tag-id="11" class="component organism">MSD</sd-pretag> and (<strong>D</strong>) <sd-pretag id="sdPretag14313348585070" parent-tag-id="12" class="component organism">MPS‐IIIA</sd-pretag> <sd-pretag id="sdPretag14313348602840" parent-tag-id="6" class="component cell">MEFs</sd-pretag> treated with <sd-pretag id="sdPretag14313349065150" parent-tag-id="173" class="component molecule">MβCD</sd-pretag>. Arrowheads and enlarged images show <sd-pretag id="sdPretag14313348984030" parent-tag-id="60" class="component molecule">cholesterol</sd-pretag> accumulation in endolysosomes of <sd-pretag id="sdPretag14313348984031" parent-tag-id="60" class="component molecule">cholesterol</sd-pretag>‐loaded WT <sd-pretag id="sdPretag14313348602841" parent-tag-id="6" class="component cell">MEFs</sd-pretag>. After treatments the rate of <sd-pretag id="sdPretag14313349401400" parent-tag-id="27" class="component subcellular">autophagosome</sd-pretag> maturation (<strong>B</strong>, <strong>E</strong>) and the transport of fluorescent <sd-tag id="sdTag509" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="dextran" ext_ids="CHEBI:52071" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="dextran" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">dextran</sd-tag> to lysosomes (<strong>C</strong>, <strong>F</strong>) were also analysed as in Figure 1. WT controls for <sd-pretag id="sdPretag14313349201000" parent-tag-id="27" class="component subcellular">autophagosome</sd-pretag> maturation and <sd-tag id="sdTag579" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="dextran" ext_ids="CHEBI:52071" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="dextran" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">dextran</sd-tag> transport experiments were performed as shown in Figure 1. (<strong>A</strong>-<strong>F</strong>) Values represent the mean±s.e.m. values of three independent experiments. <sup>*</sup><em>P</em>0.05, Student's <em>t</em>‐test: (<strong>A</strong>, <strong>C</strong>): WT versus WT+<sd-pretag id="sdPretag14313348666100" parent-tag-id="60" class="component molecule">cholesterol</sd-pretag>; (<strong>B</strong>): WT versus WT+<sd-pretag id="sdPretag14313349275940" parent-tag-id="60" class="component molecule">cholesterol</sd-pretag> for each time point; (<strong>D</strong>, <strong>F</strong>): <sd-tag id="sdTag510" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> versus <sd-tag id="sdTag513" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+<sd-tag id="sdTag516" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> and <sd-tag id="sdTag515" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> versus <sd-pretag id="sdPretag14313349118411" parent-tag-id="12" class="component organism">MPS‐IIIA</sd-pretag>+<sd-pretag id="sdPretag14313348514490" parent-tag-id="173" class="component molecule">MβCD</sd-pretag>; (<strong>E</strong>): <sd-pretag id="sdPretag14313348558510" parent-tag-id="11" class="component organism">MSD</sd-pretag> versus <sd-tag id="sdTag512" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+<sd-tag id="sdTag511" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> and <sd-pretag id="sdPretag14313348978970" parent-tag-id="12" class="component organism">MPS‐IIIA</sd-pretag> versus <sd-tag id="sdTag580" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag>+<sd-pretag id="sdPretag14313349279810" parent-tag-id="173" class="component molecule">MβCD</sd-pretag> for each time point. Scale bar: 10 μm (<strong>A</strong>, <strong>C</strong>, <strong>D</strong>, <strong>F</strong>).<graphic href="https://api.sourcedata.io/file.php?panel_id=8233"/></sd-panel></fig><fig id="1934"><title></title><label>Figure 4</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1934"/><sd-panel panel_id="5040"><b>(A)</b> <sd-tag id="sdTag97" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="Endolysosomal membranes" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">Endolysosomal membranes</sd-tag> from <sd-tag id="sdTag102" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, <sd-tag id="sdTag103" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> and WT <sd-tag id="sdTag104" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> were treated with 1% <sd-tag id="sdTag98" source="sdapi" category="entity" entity_type="molecule" role="component" text="Triton X‐114" ext_ids="CHEBI:9750" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="Triton X-100" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">Triton X‐114</sd-tag> and loaded on a <sd-tag id="sdTag109" source="sdapi" category="assay" entity_type="" role="" text="sucrose gradient" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">sucrose gradient</sd-tag>. <sd-tag id="sdTag100" source="sdapi" category="assay" entity_type="" role="" text="Immunoblots" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">Immunoblots</sd-tag> with <sd-tag id="sdTag101" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Flotillin‐1" ext_ids="O08917" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Flot1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Flotillin‐1</sd-tag> identified <sd-tag id="sdTag108" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRMs" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRMs</sd-tag> in fractions 2, 3 and 4 (arrows). The fractions at the bottom of the gradient (12 and 13) correspond to high‐density <sd-tag id="sdTag111" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="detergent soluble fractions" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">detergent <sd-pretag id="sdPretag14313257758570" parent-tag-id="124" class="component subcellular"><sd-pretag id="sdPretag14313257931730" parent-tag-id="133" class="component subcellular">soluble</sd-pretag> fractions</sd-pretag></sd-tag>, whereas the remaining ones were defined as <sd-tag id="sdTag110" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="intermediate fractions" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls=""><sd-pretag id="sdPretag14313261774570" parent-tag-id="169" class="component subcellular">intermediate</sd-pretag> fractions</sd-tag> (<sd-pretag id="sdPretag14313261196090" parent-tag-id="169" class="component subcellular">intermediate</sd-pretag>‐I: 5, 6, 7 8; <sd-pretag id="sdPretag14313261196091" parent-tag-id="169" class="component subcellular">intermediate</sd-pretag>‐II: 9, 10 and 11). The percentage of <sd-tag id="sdTag105" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Flotillin‐1" ext_ids="O08917" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Flot1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Flotillin‐1</sd-tag> in <sd-tag id="sdTag560" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRMs" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRMs</sd-tag> was calculated from the densitometric quantification of <sd-tag id="sdTag107" source="sdapi" category="assay" entity_type="" role="" text="immunoblots" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblots</sd-tag>.<graphic href="https://api.sourcedata.io/file.php?panel_id=5040"/></sd-panel><sd-panel panel_id="5041"><b>(B)</b> <sd-tag id="sdTag112" source="sdapi" category="assay" entity_type="" role="" text="Immuno‐EM" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Immuno‐EM</sd-tag> of <sd-tag id="sdTag113" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="GM1" ext_ids="CHEBI:61048" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="ganglioside GM1" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">GM1</sd-tag> lipid was carried out in WT, <sd-tag id="sdTag115" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and <sd-tag id="sdTag116" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> <sd-tag id="sdTag117" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> by staining cells with anti‐<sd-tag id="sdTag114" source="sdapi" category="entity" entity_type="protein" role="assayed" text="cholera toxin B" ext_ids="P01556" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ctxB" ext_tax_ids="243277" ext_tax_names="Vibrio cholerae O1 biovar El Tor str. N16961" ext_urls="https://www.uniprot.org/uniprot/">cholera toxin B</sd-tag> antibodies (see Materials and methods section). The number of <sd-tag id="sdTag118" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="GM1" ext_ids="CHEBI:61048" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="ganglioside GM1" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">GM1</sd-tag>‐positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT.<graphic href="https://api.sourcedata.io/file.php?panel_id=5041"/></sd-panel><sd-panel panel_id="5042"><b>(C)</b> <sd-tag id="sdTag125" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="Endolysosomal membranes" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">Endolysosomal membranes</sd-tag> from <sd-tag id="sdTag126" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, <sd-tag id="sdTag127" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> and WT <sd-tag id="sdTag128" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> were stained with <sd-tag id="sdTag122" source="sdapi" category="assay" entity_type="" role="" text="C‐laurdan" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">C‐laurdan</sd-tag> and subsequently analysed by <sd-tag id="sdTag123" source="sdapi" category="assay" entity_type="" role="" text="fluorescence spectrophotometry" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">fluorescence spectrophotometry</sd-tag> to calculate the GP value (see Materials and methods section for details). Distribution of <sd-tag id="sdTag130" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> was also measured throughout the gradient ad expressed as percentage of total <sd-tag id="sdTag561" source="sdapi" category="entity" entity_type="molecule" role="assayed" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> in raft (<sd-tag id="sdTag131" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRMs" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRMs</sd-tag>) and <sd-tag id="sdTag124" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="soluble fractions" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls=""><sd-pretag id="sdPretag14313258018730" parent-tag-id="133" class="component subcellular">soluble</sd-pretag> fractions</sd-tag>.<graphic href="https://api.sourcedata.io/file.php?panel_id=5042"/></sd-panel><sd-panel panel_id="5043"><b>(D)</b> Equal aliquots from either <sd-tag id="sdTag132" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRMs" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRMs</sd-tag> or <sd-tag id="sdTag133" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="soluble fractions" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">soluble fractions</sd-tag> were pooled, the protein content determined and displayed as percentage of total protein in <sd-tag id="sdTag134" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRM" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRM</sd-tag> and <sd-tag id="sdTag135" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="soluble" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">soluble</sd-tag> gradient regions.<graphic href="https://api.sourcedata.io/file.php?panel_id=5043"/></sd-panel><sd-panel panel_id="5044"><b>(E)</b> <sd-tag id="sdTag144" source="sdapi" category="assay" entity_type="" role="" text="Immunoblotting" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">Immunoblotting</sd-tag> profiles of the transferrin receptor and <sd-tag id="sdTag145" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LAMP1" ext_ids="P11438" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Lamp1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">LAMP1</sd-tag> in the <sd-tag id="sdTag146" source="sdapi" category="assay" entity_type="" role="" text="sucrose gradient" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">sucrose gradient</sd-tag>. Values represent the mean±s.e.m. values of three experiments (<b>A</b>-<b>D</b>). <sup>*</sup><i>P</i>0.05, Student's <i>t</i>‐test: (<b>A</b>-<b>C</b>): WT versus <sd-tag id="sdTag147" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and WT versus <sd-tag id="sdTag148" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag>; (<b>D</b>): WT versus <sd-tag id="sdTag149" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and WT versus <sd-tag id="sdTag150" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> for each fraction. Scale bar: 0.3 μm (<b>B</b>).<graphic href="https://api.sourcedata.io/file.php?panel_id=5044"/></sd-panel></fig><fig id="1935"><title></title><label>Figure 5</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1935"/><sd-panel panel_id="5045"><b>(A)</b> <sd-tag id="sdTag161" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag>, <sd-tag id="sdTag162" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> and <sd-tag id="sdTag163" source="sdapi" category="entity" entity_type="protein" role="assayed" text="syntaxin 7" ext_ids="O70439" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Stx7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">syntaxin 7</sd-tag> distributions in <sd-tag id="sdTag164" source="sdapi" category="entity" entity_type="subcellular" role="component" text="endo‐lysosomal membranes" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">endo‐lysosomal membranes</sd-tag> from <sd-tag id="sdTag177" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, <sd-tag id="sdTag165" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPSIIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPSIIIA</sd-tag> and WT <sd-tag id="sdTag178" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> was evaluated by <sd-tag id="sdTag166" source="sdapi" category="assay" entity_type="" role="" text="immonoblotting" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">immonoblotting</sd-tag> analysis of <sd-tag id="sdTag167" source="sdapi" category="assay" entity_type="" role="" text="gradient fractions" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">gradient fractions</sd-tag>. To simplify the analysis the <sd-tag id="sdTag179" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRM" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRM</sd-tag>, the <sd-tag id="sdTag169" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="intermediate‐I" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">intermediate‐I</sd-tag>, the <sd-tag id="sdTag170" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="intermediate‐II" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">intermediate‐II</sd-tag> and <sd-tag id="sdTag168" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="soluble fractions" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">soluble fractions</sd-tag> of the gradient were pooled separately and then subjected to <sd-tag id="sdTag171" source="sdapi" category="assay" entity_type="" role="" text="immunoblotting" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblotting</sd-tag>. <sd-tag id="sdTag172" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="SNARE" ext_ids="GO:0031201" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="SNARE complex" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">SNARE</sd-tag> distribution was also analysed after loading WT cells with <sd-tag id="sdTag180" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> and after <sd-tag id="sdTag173" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> treatment. In <sd-tag id="sdTag181" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and <sd-tag id="sdTag182" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> <sd-tag id="sdTag183" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>, all analysed <sd-tag id="sdTag174" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="SNAREs" ext_ids="GO:0031201" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="SNARE complex" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">SNAREs</sd-tag> abnormally accumulate in <sd-tag id="sdTag184" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRMs" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRMs</sd-tag> of <sd-tag id="sdTag175" source="sdapi" category="entity" entity_type="subcellular" role="component" text="lysosomal membranes" ext_ids="GO:0005765" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="lysosomal membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">lysosomal membranes</sd-tag>. <sd-tag id="sdTag185" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="Cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">Cholesterol</sd-tag> modulation results in a change of <sd-tag id="sdTag562" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="SNARE" ext_ids="GO:0031201" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="SNARE complex" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">SNARE</sd-tag> distribution.<graphic href="https://api.sourcedata.io/file.php?panel_id=5045"/></sd-panel><sd-panel panel_id="5046"><b>(B)</b> The percentage of each analysed <sd-tag id="sdTag187" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="SNARE" ext_ids="GO:0031201" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="SNARE complex" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">SNARE</sd-tag> observed in <sd-tag id="sdTag188" source="sdapi" category="entity" entity_type="subcellular" role="component" text="DRM" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRM</sd-tag> fractions was quantified from blots (ImageJ <sd-tag id="sdTag189">densitometry analysis</sd-tag>) and displayed as relative amount versus WT.<graphic href="https://api.sourcedata.io/file.php?panel_id=5046"/></sd-panel><sd-panel panel_id="5047"><b>(C, E)</b> <sd-tag id="sdTag210" source="sdapi" category="assay" entity_type="" role="" text="Immunoblots" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">Immunoblots</sd-tag> and relative quantification showing <sd-tag id="sdTag211" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="SNARE" ext_ids="GO:0031201" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="SNARE complex" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">SNARE</sd-tag> protein levels along with <sd-tag id="sdTag212" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LAMP1" ext_ids="P11438" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Lamp1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">LAMP1</sd-tag> protein levels in (<b>C</b>) <sd-tag id="sdTag213" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="endolysosomal" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">endolysosomal</sd-tag> membranes and (<b>E</b>) total cell lysates from WT (untreated and <sd-tag id="sdTag214" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> treated) and <sd-tag id="sdTag215" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> <sd-tag id="sdTag216" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> (untreated and <sd-tag id="sdTag217" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> treated). In the graphs, the protein levels were displayed as relative amount versus WT.<graphic href="https://api.sourcedata.io/file.php?panel_id=5047"/></sd-panel><sd-panel panel_id="5048"><b>(D)</b> WT and <sd-tag id="sdTag222" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> <sd-tag id="sdTag223" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> transfected with <sd-tag id="sdTag228" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>-<sd-tag id="sdTag225" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag> were stained with anti‐<sd-tag id="sdTag229" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag> for <sd-tag id="sdTag227" source="sdapi" category="assay" entity_type="" role="" text="immuno‐EM" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">immuno‐EM</sd-tag>. The enlarged image shows internalization of <sd-tag id="sdTag564" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>-<sd-tag id="sdTag563" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag> particles (arrow).<graphic href="https://api.sourcedata.io/file.php?panel_id=5048"/></sd-panel><sd-panel panel_id="5049"><b>(F)</b> <sd-tag id="sdTag230" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Syntaxin 5" ext_ids="Q8K1E0" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Stx5" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Syntaxin 5</sd-tag>, <sd-tag id="sdTag231" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Sec22" ext_ids="Q8BH47///Q8BXT9///O08547" norm_text="" ext_dbs="Uniprot///Uniprot///Uniprot" in_caption="True" ext_names="Sec22a///Sec22c///Sec22b" ext_tax_ids="10090///10090///10090" ext_tax_names="Mus musculus///Mus musculus///Mus musculus" ext_urls="https://www.uniprot.org/uniprot////https://www.uniprot.org/uniprot////https://www.uniprot.org/uniprot/">Sec22</sd-tag> and <sd-tag id="sdTag232" source="sdapi" category="entity" entity_type="protein" role="assayed" text="SNAP23" ext_ids="O09044" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Snap23" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">SNAP23</sd-tag> distribution in total membrane derived from control WT and <sd-tag id="sdTag234" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> <sd-tag id="sdTag235" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>. <sd-tag id="sdTag236" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Syntaxin 5" ext_ids="Q8K1E0" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Stx5" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Syntaxin 5</sd-tag> <sd-tag id="sdTag233" source="sdapi" category="assay" entity_type="" role="" text="immunoblot" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblot</sd-tag> shows two bands (<sup>*</sup>, 35 kDa and <sup>**</sup>, 42 kDa) corresponding to the two isoforms of the protein.<graphic href="https://api.sourcedata.io/file.php?panel_id=5049"/></sd-panel><sd-panel panel_id="5050"><b>(G)</b> Distribution profile of <sd-tag id="sdTag240" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Rab7" ext_ids="P51150" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Rab7a" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Rab7</sd-tag> in WT and <sd-tag id="sdTag243" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> <sd-tag id="sdTag242" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="lysosomal membranes" ext_ids="GO:0005765" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="lysosomal membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">lysosomal membranes</sd-tag>. Values represent the mean±s.e.m. values of three independent experiments (<b>B</b>, <b>C</b>, <b>E</b>). <sup>*</sup><i>P</i>0.05, Student's <i>t</i>‐test: (<b>B</b>): WT versus <sd-tag id="sdTag565" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, WT versus <sd-pretag id="sdPretag14313355679170" parent-tag-id="507">MPS‐IIIA</sd-pretag>, WT versus WT+<sd-pretag id="sdPretag14313286465960" parent-tag-id="321" class="component molecule">cholesterol</sd-pretag>, <sd-tag id="sdTag247" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> versus <sd-tag id="sdTag566" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+<sd-pretag id="sdPretag14313287174180" parent-tag-id="335" class="component molecule">MβCD</sd-pretag>, and <sd-pretag id="sdPretag14313355223420" parent-tag-id="507">MPS‐IIIA</sd-pretag> versus <sd-pretag id="sdPretag14313287292100" parent-tag-id="335" class="component molecule">MβCD</sd-pretag> for each analysed SNARE; (<b>C</b>, <b>E</b>): WT versus <sd-tag id="sdTag254" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, WT versus WT+<sd-pretag id="sdPretag14313286440650" parent-tag-id="321" class="component molecule">cholesterol</sd-pretag> and <sd-tag id="sdTag255" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> versus <sd-tag id="sdTag567" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+<sd-pretag id="sdPretag14313287360240" parent-tag-id="335" class="component molecule">MβCD</sd-pretag> for each analysed protein. Scale bar: 0.3 μm (<b>D</b>).<graphic href="https://api.sourcedata.io/file.php?panel_id=5050"/></sd-panel></fig><fig id="1936"><title></title><label>Figure 6</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1936"/><sd-panel panel_id="5051"><b>(A)</b> <sd-tag id="sdTag262" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="SDS" ext_ids="CHEBI:8984" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="sodium dodecyl sulfate" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">SDS</sd-tag>‐resistant complexes containing <sd-tag id="sdTag264" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> were detected by <sd-tag id="sdTag266" source="sdapi" category="assay" entity_type="" role="" text="immunoblotting" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblotting</sd-tag> analysis of nonboiled samples corresponding to total, detergent insoluble (<sd-tag id="sdTag268" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRM" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRM</sd-tag>) and detergent <sd-tag id="sdTag270" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="soluble" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">soluble</sd-tag> (Sol.) <sd-tag id="sdTag263" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="endo‐lysosomal membrane" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">endo‐lysosomal membrane</sd-tag> fractions derived from <sd-tag id="sdTag272" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and WT <sd-tag id="sdTag274" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>. The <sd-tag id="sdTag276" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="SDS" ext_ids="CHEBI:8984" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="sodium dodecyl sulfate" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">SDS</sd-tag>‐resistant complexes were also visualized after loading WT <sd-tag id="sdTag278" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> with <sd-tag id="sdTag280" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> and after treating <sd-tag id="sdTag282" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> <sd-tag id="sdTag284" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> with <sd-tag id="sdTag286" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag>. <sd-tag id="sdTag288" source="sdapi" category="assay" entity_type="" role="" text="Immunoblots" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">Immunoblots</sd-tag> revealed the presence of low molecular weight complexes (<sup>*</sup>, 50-60 kDa) and high molecular weight complexes (<sup>**</sup>, &gt;80 kDa). The percentage of <sd-tag id="sdTag290" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> in <sd-tag id="sdTag292" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="SDS" ext_ids="CHEBI:8984" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="sodium dodecyl sulfate" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">SDS</sd-tag>‐resistant complexes in total <sd-tag id="sdTag294" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="endolysosomal" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">endolysosomal</sd-tag> membranes (bottom‐left chart) and the amount of <sd-tag id="sdTag296" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag>‐containing <sd-tag id="sdTag298" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="SDS" ext_ids="CHEBI:8984" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="sodium dodecyl sulfate" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">SDS</sd-tag>‐resistant complexes in <sd-tag id="sdTag300" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="DRM" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">DRM</sd-tag> and <sd-tag id="sdTag302" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="soluble fractions" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">soluble fractions</sd-tag> (bottom‐right chart) were calculated by the densitometric quantification of the correspondent <sd-tag id="sdTag306" source="sdapi" category="assay" entity_type="" role="" text="immunoblots" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunoblots</sd-tag> (ImageJ). Values represent the mean±s.e.m. values of three independent measurements. <sup>*</sup><i>P</i>0.05, Student's <i>t</i>‐test: WT versus <sd-tag id="sdTag308" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, WT versus WT+<sd-tag id="sdTag310" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> and <sd-tag id="sdTag314" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> versus <sd-tag id="sdTag568" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+<sd-tag id="sdTag316" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> (bottom‐left chart); WT versus <sd-tag id="sdTag318" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, WT versus WT+<sd-tag id="sdTag320" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> and <sd-tag id="sdTag324" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> versus <sd-tag id="sdTag569" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>+<sd-tag id="sdTag326" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> for each fraction (bottom‐right chart).<graphic href="https://api.sourcedata.io/file.php?panel_id=5051"/></sd-panel><sd-panel panel_id="5052"><b>(B)</b> <sd-tag id="sdTag328" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Syntaxin 7" ext_ids="O70439" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Stx7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Syntaxin 7</sd-tag> and <sd-tag id="sdTag330" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag> were <sd-tag id="sdTag329" source="sdapi" category="assay" entity_type="" role="" text="co‐immunoprecipitated" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">co‐immunoprecipitated</sd-tag> with <sd-tag id="sdTag332" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> using anti‐<sd-tag id="sdTag570" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> antibodies in WT (untreated or <sd-tag id="sdTag333" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> treated) and in <sd-tag id="sdTag334" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> (not treated or <sd-tag id="sdTag335" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> treated) <sd-tag id="sdTag336" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>. The amount of <sd-tag id="sdTag337" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> precipitated in each cell line is also shown.<graphic href="https://api.sourcedata.io/file.php?panel_id=5052"/></sd-panel><sd-panel panel_id="5053"><b>(C)</b> <sd-tag id="sdTag340" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="SDS" ext_ids="CHEBI:8984" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="sodium dodecyl sulfate" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">SDS</sd-tag>‐resistant complexes are decorated by anti‐<sd-tag id="sdTag341" source="sdapi" category="entity" entity_type="protein" role="assayed" text="syntaxin 7" ext_ids="O70439" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Stx7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">syntaxin 7</sd-tag> antibodies in total <sd-tag id="sdTag339" source="sdapi" category="entity" entity_type="subcellular" role="component" text="endolysosomal membrane" ext_ids="GO:0036020" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="endolysosome membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">endolysosomal membrane</sd-tag> fraction from WT and <sd-tag id="sdTag342" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> <sd-tag id="sdTag343" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>.<graphic href="https://api.sourcedata.io/file.php?panel_id=5053"/></sd-panel><sd-panel panel_id="5054"><b>(D)</b> <sd-tag id="sdTag349" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="Membrane" ext_ids="GO:0005886" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="plasma membrane" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">Membrane</sd-tag>‐associated <sd-tag id="sdTag347" source="sdapi" category="entity" entity_type="protein" role="assayed" text="α‐SNAP" ext_ids="Q9DB05" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Napa" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">α‐SNAP</sd-tag> and its release in the <sd-tag id="sdTag348" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="cytosol" ext_ids="GO:0005829" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="cytosol" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">cytosol</sd-tag> were evaluated by <sd-tag id="sdTag350" source="sdapi" category="assay" entity_type="" role="" text="western blot" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&amp;conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">western blot</sd-tag> analysis on total cell lysates (total), intracellular membranes recovered after centrifugation from a post‐nuclear supernatant fraction (membrane associated) and cell lysates devoid of membranes (cytosolic released) derived from <sd-tag id="sdTag351" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> (untreated or <sd-tag id="sdTag352" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> treated) and WT (untreated or <sd-tag id="sdTag353" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> treated) <sd-tag id="sdTag354" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>.<graphic href="https://api.sourcedata.io/file.php?panel_id=5054"/></sd-panel></fig><fig id="1937"><title></title><label>Figure 7</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1937"/><sd-panel panel_id="5055"><b>(A)</b> <sd-tag id="sdTag355" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag>, <sd-tag id="sdTag356" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> and WT <sd-tag id="sdTag357" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> were subjected to a triple labelling with anti‐<sd-tag id="sdTag358" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag>, anti‐<sd-tag id="sdTag359" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> and anti‐<sd-tag id="sdTag360" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LAMP1" ext_ids="P11438" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Lamp1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">LAMP1</sd-tag> antibodies. The merges between <sd-tag id="sdTag363" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag> and <sd-tag id="sdTag364" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> (double merges in yellow) and between <sd-tag id="sdTag571" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag>, <sd-tag id="sdTag572" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> and <sd-tag id="sdTag365" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LAMP1" ext_ids="P11438" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Lamp1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">LAMP1</sd-tag> (triple merges in white) are shown (see also enlarged images showing the extent of co‐localization in different regions of the cells). The <sd-tag id="sdTag366" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag>-<sd-tag id="sdTag368" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> co‐localization was quantified in 15 different cells and displayed as % of <sd-tag id="sdTag573" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> co‐localizing with <sd-tag id="sdTag369" source="sdapi" category="entity" entity_type="protein" role="assayed" text="VAMP7" ext_ids="P70280" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vamp7" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">VAMP7</sd-tag> (means±s.e.m.).<graphic href="https://api.sourcedata.io/file.php?panel_id=5055"/></sd-panel><sd-panel panel_id="5056"><b>(B)</b> Co‐localization of <sd-tag id="sdTag372" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> with <sd-tag id="sdTag371" source="sdapi" category="entity" entity_type="protein" role="assayed" text="epsinR" ext_ids="Q99KN9" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Clint1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">epsinR</sd-tag> was quantified by double‐labelling experiments in <sd-tag id="sdTag373" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> and <sd-tag id="sdTag374" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MPS‐IIIA" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MPS‐IIIA</sd-tag> (untreated and <sd-tag id="sdTag375" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> treated) and in control WT (untreated and <sd-tag id="sdTag376" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> treated) <sd-tag id="sdTag377" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag>. The chart displays merge values (means±s.e.m.) that represent the percentage of <sd-tag id="sdTag378" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> co‐localizing with <sd-tag id="sdTag379" source="sdapi" category="entity" entity_type="protein" role="assayed" text="epsinR" ext_ids="Q99KN9" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Clint1" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">epsinR</sd-tag> measured in 15 different cells.<graphic href="https://api.sourcedata.io/file.php?panel_id=5056"/></sd-panel><sd-panel panel_id="5057"><p><span>(</span><span>C</span><span>) <sd-tag id="sdTag537" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> trafficking was monitored by <sd-tag id="sdTag538" source="sdapi" category="assay" entity_type="" role="" text="FRAP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">FRAP</sd-tag> analysis in WT (untreated and <sd-tag id="sdTag539" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="cholesterol" ext_ids="CHEBI:16113" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="cholesterol" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">cholesterol</sd-tag> treated) and <sd-tag id="sdTag542" source="sdapi" category="entity" entity_type="organism" role="experiment" text="MSD" ext_ids="10090" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mus musculus" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">MSD</sd-tag> (untreated and <sd-tag id="sdTag540" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MβCD" ext_ids="CHEBI:495055" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="beta-cyclodextrin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MβCD</sd-tag> treated) <sd-tag id="sdTag541" source="sdapi" category="entity" entity_type="cell_line" role="component" text="MEFs" ext_ids="CL:2000042" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="embryonic fibroblast" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://identifiers.org/cellosaurus:">MEFs</sd-tag> transfected with <sd-tag id="sdTag543" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>-<sd-tag id="sdTag544" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Vti1b" ext_ids="O88384" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="Vti1b" ext_tax_ids="10090" ext_tax_names="Mus musculus" ext_urls="https://www.uniprot.org/uniprot/">Vti1b</sd-tag> (see Materials and methods section for details). <sd-tag id="sdTag545" source="sdapi" category="assay" entity_type="" role="" text="FRAP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">FRAP</sd-tag> data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells.</span></p><graphic href="https://api.sourcedata.io/file.php?panel_id=5057"/></sd-panel></fig><fig id="1938"><title></title><label>Figure 8</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1938"/></fig></article>