Figure 4C - F ) C ) COL1A1 and COL1A2 expression in the ER of SEC16B patient and control fibroblasts assessed by western blotting . KDEL , Calnexin , BIP and PDI , all ER - resident proteins were used as ER markers . E ) Golgi complex was isolated according to the method described in Current Protocols in Cell Biology , Unit 3 . 9 from healthy control cells and patient derived fibroblasts . Subsequently , COL1A1 and COL1A2 expression in the Golgi complex of SEC16B patient and control fibroblasts were determined by western blotting . GM130 ( Golgi - resident protein ) was used as marker for Golgi complex . All cells were treated with ascorbic acid 0 . 05 mg / ml for 20 hours before the Golgi complex was isolated . D , F ) Quantification of the bands that was done with Bio - Rad Image Lab 6 . 1 software to calculate the expression levels . COL1A1 and COL1A2 band intensities were either normalized to ( D ) calnexin or ( F ) GM130 . The normalized intensities were reported as fold change patient to control cells . Columns represent the mean of three ( D ) or two ( F ) independent experiments ( raw values : 0 . 27 and 0 . 53 for COL1A1 and 0 . 29 , 0 . 21 for COL1A2 ) respectively . Biological replicates were performed ; bars , standard error of the mean ( SEM ) ; * significant ( p < 0 . 05 ) differences obtained by using unpaired two - tailed t

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C-F) C) COL1A1 and COL1A2 expression in the ER of SEC16B patient and control fibroblasts assessed by western blotting. KDEL, Calnexin, BIP and PDI, all ER-resident proteins were used as ER markers. E) Golgi complex was isolated according to the method described in Current Protocols in Cell Biology, Unit 3.9 from healthy control cells and patient derived fibroblasts. Subsequently, COL1A1 and COL1A2 expression in the Golgi complex of SEC16B patient and control fibroblasts were determined by western blotting. GM130 (Golgi-resident protein) was used as marker for Golgi complex. All cells were treated with ascorbic acid 0.05 mg/ml for 20 hours before the Golgi complex was isolated. D, F) Quantification of the bands that was done with Bio-Rad Image Lab 6.1 software to calculate the expression levels. COL1A1 and COL1A2 band intensities were either normalized to (D) calnexin or (F) GM130. The normalized intensities were reported as fold change patient to control cells. Columns represent the mean of three (D) or two (F) independent experiments (raw values: 0.27 and 0.53 for COL1A1 and 0.29, 0.21 for COL1A2) respectively. Biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two-tailed t test


Figure 5A ) Patient and control fibroblasts were plated on glass cover slips . After 24 hours , cells were fixed and stained for SEC31 to label ERES . The scale bars are 10 µm . B ) Quantification of the number of ERES per area from cells processed . The values are from four independent experiments , biological replicates . 71 cell per experiment were acquired . Bars are means ± standard error of the mean ( SEM ) ; * significant ( p < 0 . 05 ) differences obtained by using a paired two - tailed t - test . C ) Patient and control fibroblasts were electroporated with plasmids encoding mCherry - tagged Mannosidase II ( ManII ) as part of a RUSH construct . After 24 hours , cells were fixed ( T0 ) or treated with 40 mM biotin for 20 minutes ( T20 ) followed by fixation and immunostaining for GM130 to label the Golgi complex . The scale bars are 10 µm . D ) quantification of 59 to 81 cells from three independent RUSH experiments ( biological replicates ) . Biological replicates were performed . Central band represents the median . Whiskers are minimum and maximum values . Outliers are defined as greater than 1 . 5 fold of upper quartiles . * * highly significant ( p < 0 . 005 ) differences obtained by unpaired two tailed t -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Patient and control fibroblasts were plated on glass cover slips. After 24 hours, cells were fixed and stained for SEC31 to label ERES. The scale bars are 10 µm. B) Quantification of the number of ERES per area from cells processed. The values are from four independent experiments, biological replicates. 71 cell per experiment were acquired. Bars are means ± standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using a paired two-tailed t-test.C) Patient and control fibroblasts were electroporated with plasmids encoding mCherry-tagged Mannosidase II (ManII) as part of a RUSH construct. After 24 hours, cells were fixed (T0) or treated with 40 mM biotin for 20 minutes (T20) followed by fixation and immunostaining for GM130 to label the Golgi complex. The scale bars are 10 µm. D) quantification of 59 to 81 cells from three independent RUSH experiments (biological replicates). Biological replicates were performed. Central band represents the median. Whiskers are minimum and maximum values. Outliers are defined as greater than 1.5 fold of upper quartiles.** highly significant (p < 0.005) differences obtained by unpaired two tailed t-test


Figure 6A ) Micrograph of thin - section electron microscopy for fibroblasts from SEC16B mutant patient cells and healthy control cells . Arrows point to rER , arrowheads to MVBs / MLBs , star indicates autophagosome . Scale bar is 1 µm . Summary of the number of the autophagosomes . C ) Western blotting anaylsis of autophagy marker LC3B . D ) Quantification of the bands . Columns represent the mean of three independent experiments , biological replicates were performed ; bars , standard error of the mean ( SEM ) ; * significant ( p < 0 . 05 ) differences obtained by using unpaired two tailed t - test . E ) 24 hours after plating of patient and control fibroblasts , cells were treated with DMSO , 10mM thapsigargin , or 1 mg / ml tunicamycin for 6 hours . Cells were lysed and immunoblotted for XBP1s as a marker for ER stress and for vinculin to ensure equal loading . F ) Western blotting anaylsis of apoptosis marker cleaved caspase 3 show enhanced expression in patient cells when compared to healthy control . A representative experiment out of three independent experiments is shown . Biological replicates were performed . G ) Quantification of the bands . All band intensities were normalized to those of Actin and reported as fold change patient to control cells . Columns represent the mean of three independent experiments , biological replicates were performed ; bars , standard error of the mean ( SEM ) ; * significant ( p < 0 . 05 ) differences obtained by using unpaired two tailed t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Micrograph of thin-section electron microscopy for fibroblasts from SEC16B mutant patient cells and healthy control cells. Arrows point to rER, arrowheads to MVBs/MLBs, star indicates autophagosome. Scale bar is 1 µm. Summary of the number of the autophagosomes.C) Western blotting anaylsis of autophagy marker LC3B. D) Quantification of the bands. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two tailed t-test.E) 24 hours after plating of patient and control fibroblasts, cells were treated with DMSO, 10mM thapsigargin, or 1 mg/ml tunicamycin for 6 hours. Cells were lysed and immunoblotted for XBP1s as a marker for ER stress and for vinculin to ensure equal loading.F) Western blotting anaylsis of apoptosis marker cleaved caspase 3 show enhanced expression in patient cells when compared to healthy control. A representative experiment out of three independent experiments is shown. Biological replicates were performed. G) Quantification of the bands. All band intensities were normalized to those of Actin and reported as fold change patient to control cells. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two tailed t-test.


Figure 7Patient cells were either transfected with pCDNA3 empty vector ( empty vector ) or pcDNA3 which encodes wt SEC16B ( TF ) . Cells were then selected with puromycin and single colonies were picked up and expanded . Established single colonies were used . B ) Immunofluorescence labeling of the control and the patient fibroblasts for COL1A1 marker proα1 ( I ) and rough ER marker KDEL . The staining showed that while COL1A1 is accumulated in the ER of patient cells transfected with empty vector , it is not accumulated in the ER of the patient cells transfected with SEC16B wt . Scale bar is 25 µmPatient cells were either transfected with pCDNA3 empty vector ( empty vector ) or pcDNA3 which encodes wt SEC16B ( TF ) . Cells were then selected with puromycin and single colonies were picked up and expanded . Established single colonies were used . D ) LC3B showed reduced expression in the TF SEC16B mutant cells ( similar to expression in healthy control Fig . 5B ) when compared to empty vector transfected patient cells . E ) Quantification of the bands . All band intensities were normalized to those of Actin and reported as fold change patient to control cells . Columns represent the mean of three independent experiments , biological replicates were performed ; bars , standard error of the mean ( SEM ) ; * * highly significant ( p < 0 . 001 differences obtained by using unpaired two tailed t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Patient cells were either transfected with pCDNA3 empty vector (empty vector) or pcDNA3 which encodes wt SEC16B (TF). Cells were then selected with puromycin and single colonies were picked up and expanded. Established single colonies were used. B) Immunofluorescence labeling of the control and the patient fibroblasts for COL1A1 marker proα1 (I) and rough ER marker KDEL. The staining showed that while COL1A1 is accumulated in the ER of patient cells transfected with empty vector, it is not accumulated in the ER of the patient cells transfected with SEC16B wt. Scale bar is 25 µmPatient cells were either transfected with pCDNA3 empty vector (empty vector) or pcDNA3 which encodes wt SEC16B (TF). Cells were then selected with puromycin and single colonies were picked up and expanded. Established single colonies were used. D) LC3B showed reduced expression in the TF SEC16B mutant cells (similar to expression in healthy control Fig. 5B) when compared to empty vector transfected patient cells. E) Quantification of the bands. All band intensities were normalized to those of Actin and reported as fold change patient to control cells. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); **highly significant (p < 0.001 differences obtained by using unpaired two tailed t-test.


Figure 8A ) HeLa cells were transfected with plasmids encoding GFP - tagged SEC16B - R142W . After 24 hours , cells were fixed and immunostained against SEC13 to label ERES . Green color shows SEC16B - R142W expression , red colors shows SEC13 expression and yellow color shows the overlap of the two colors . The scale bars are 10 µm . D ) HepG2 cells were transfected with the indicated siRNA . After 48h , cells were transfected with plasmids encoding signal sequence GFP ( ssGFP ) as part of a RUSH construct . After 24 hours , cells were treated with 40 mM biotin for 15 minutes followed by fixation and immunostaining for GM130 to label the Golgi complex . The scale bars are 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) HeLa cells were transfected with plasmids encoding GFP-tagged SEC16B-R142W. After 24 hours, cells were fixed and immunostained against SEC13 to label ERES. Green color shows SEC16B-R142W expression, red colors shows SEC13 expression and yellow color shows the overlap of the two colors. The scale bars are 10 µm.D) HepG2 cells were transfected with the indicated siRNA. After 48h, cells were transfected with plasmids encoding signal sequence GFP (ssGFP) as part of a RUSH construct. After 24 hours, cells were treated with 40 mM biotin for 15 minutes followed by fixation and immunostaining for GM130 to label the Golgi complex. The scale bars are 10 µm.


Figure 1Ability of the SARS - CoV - 2 DNA vaccine OC - 2 . 4 to induce antibodies against S protein of the huCoV - 19 / WH01 ( B ) and Beta variants ( D ) Ability of the SARS - CoV - 2 DNA vaccine OC - 2 . 4 to induce antibodies against S protein the ability to neutralize these viruses in vitro ( C and E ) . Also shown is the ability of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 to prime and boost antibodies to S protein ( huCoV - 19 / WH01 ) following priming with a recombinant S protein in adjuvant ( F ) Also shown is the ability of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 to prime the ability of these antibodies to neutralize the Delta and Omicron variants in vitro ( G ) . Finally , three doses of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 induce antibodies that cross react with S proteins from the huCoV - 19 / WH01 , Beta , and Delta variants in mice ( H ) , four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro ( I ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein of the huCoV-19/WH01 (B) and Beta variants (D)Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein the ability to neutralize these viruses in vitro (C and E).Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime and boost antibodies to S protein (huCoV-19/WH01) following priming with a recombinant S protein in adjuvant (F)Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime the ability of these antibodies to neutralize the Delta and Omicron variants in vitro (G).Finally, three doses of the universal SARS-CoV-2 DNA vaccine OC-2.4 induce antibodies that cross react with S proteins from the huCoV-19/WH01, Beta, and Delta variants in mice (H),four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro (I).


Figure 2A - D ) T cell responses as determined by ELISpot in Balb / c mice immunized three times with either spike protein in QS21 adjuvant ( rS / QS21 ; A ) , 50µg universal SARS - CoV - 2 vaccine OC - 2 . 4 ( B ) , primed with rS / QS21 and boosted with two doses of OC - 2 . 4 ( C ) , or empty pVAX plasmid ( D ) , using in vivo electroporation . Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens . Data has been given as the number of IFNγ producing ( spot forming ) cells ( SFCs ) per million splenocytes . Each color indicate an individual mice . E - H ) Also shown is analysis of T cell responses in New Zeeland White rabbits , which were immunized four times with either vehicle only ( E ) , or 84µg ( F ) or 840 µg ( G ) universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 using in vivo electroporation . Peripheral blood mononuclear cells ( PBMCs ) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens . Data has been given as the number of IFNγ SFCs per million PBMCs . The dotted line indicates the 50 SFC cut off . Also shown ( H ) is the cumulated IFNγ SFCs per million PBMCs to the RBD , M and N peptide pools added together for each group of immunized rabbits ( 84µg DNA , 840µg DNA , and vehicle only ) at each time point ( day ) . The numbers on the x - axis of graphs A ) to D ) indicates the number of the respective peptide pool ( WH1 variant ) covering a part of the indicated protein .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-D) T cell responses as determined by ELISpot in Balb/c mice immunized three times with either spike protein in QS21 adjuvant (rS/QS21; A), 50µg universal SARS-CoV-2 vaccine OC-2.4 (B), primed with rS/QS21 and boosted with two doses of OC-2.4 (C), or empty pVAX plasmid (D), using in vivo electroporation. Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ producing (spot forming) cells (SFCs) per million splenocytes. Each color indicate an individual mice.E-H) Also shown is analysis of T cell responses in New Zeeland White rabbits, which were immunized four times with either vehicle only (E), or 84µg (F) or 840 µg (G) universal SARS-CoV-2 DNA vaccine OC-2.4 using in vivo electroporation. Peripheral blood mononuclear cells (PBMCs) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ SFCs per million PBMCs. The dotted line indicates the 50 SFC cut off. Also shown (H) is the cumulated IFNγ SFCs per million PBMCs to the RBD, M and N peptide pools added together for each group of immunized rabbits (84µg DNA, 840µg DNA, and vehicle only) at each time point (day). The numbers on the x-axis of graphs A) to D) indicates the number of the respective peptide pool (WH1 variant) covering a part of the indicated protein.


Figure 3Three doses of respective vaccine fully or partially protected the mice against severe disease as determined by histological scoring of brochial and alveolar lung tissues ( b ) Three doses of respective vaccine fully or partially protected the mice against severe disease as percent weight loss ( c ) . D ) Three doses of respective vaccine also protect against viral replication in the nose , lungs and spleen . Values have been given as cycle times ( CT ) , where lower values indicate a higher viral load . Statistical comparisons in the graph are shown with lines with one asterisc indicating p < 0 , 05 and two asterics p < 0 ,

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Figure 3Three doses of respective vaccine fully or partially protected the mice against severe disease as determined by histological scoring of brochial and alveolar lung tissues (b)Three doses of respective vaccine fully or partially protected the mice against severe disease as percent weight loss (c).D) Three doses of respective vaccine also protect against viral replication in the nose, lungs and spleen. Values have been given as cycle times (CT), where lower values indicate a higher viral load. Statistical comparisons in the graph are shown with lines with one asterisc indicating p<0,05 and two asterics p<0,01


Figure 1 ( A ) Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter . Total RNA was isolated from these primary cells and primary AECs to perform chip hybridization and microarray analysis . The profile of EphA2 expression in different cells is indicated . ( B ) Murine B cells and T cells were purified from spleens using a cell sorter , total RNA was isolated from murine B cells , T cells , BMDCs , BMDMs , AMs and AECs . Shown is mean ± SD . Data represent three independent biological replicates . The profile of EphA2 expression in different cells is indicated . ( C ) Immunoblot analysis of EphA2 and β - actin in BMDCs , BMDMs , AMs , and AECs .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells and primary AECs to perform chip hybridization and microarray analysis.The profile of EphA2 expression in different cells is indicated. (B) Murine B cells and T cells were purified from spleens using a cell sorter, total RNA was isolated from murine B cells, T cells, BMDCs, BMDMs, AMs and AECs. Shown is mean ± SD. Data represent three independent biological replicates.The profile of EphA2 expression in different cells is indicated. (C) Immunoblot analysis of EphA2 and β-actin in BMDCs, BMDMs, AMs, and AECs.


Figure 2 ( A - D ) ELISA of IL - 1β , IL - 18 , IFN - β and IL - 6 in supernatant of AEC cells , which were stimulated for 16 h with RNA virus reovirus ( MOI = 5 ) or DNA virus HSV - 1 ( MOI = 5 ) . n = 3 primary samples . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . Mock indicated unstimulated cells . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . NS means no statistical difference . Data represent three independent biological replicates . ( E ) Immunoblot analysis of IL - 1β and caspase - 1 in AECs cell lysate or cell supernatant , β - actin served as a loading control throughout . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . Mock indicated unstimulated cells . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . NS means no statistical difference . Data represent three independent biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-D) ELISA of IL-1β, IL-18, IFN-β and IL-6 in supernatant of AEC cells, which were stimulated for 16 h with RNA virus reovirus (MOI=5) or DNA virus HSV-1 (MOI=5). n = 3 primary samples. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. Mock indicated unstimulated cells. **P <0.01 and ***P<0.001 (unpaired t test). NS means no statistical difference. Data represent three independent biological replicates.(E) Immunoblot analysis of IL-1β and caspase-1 in AECs cell lysate or cell supernatant, β-actin served as a loading control throughout. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. Mock indicated unstimulated cells. **P <0.01 and ***P<0.001 (unpaired t test). NS means no statistical difference. Data represent three independent biological replicates.


Figure 3 ( A - B ) ELISA of IL - 1β and IL - 18 in BALF samples were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice ( n = 3 per strain ) on day 1 and day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates . Cell number of BALF samples were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice ( n = 3 per strain ) on day 1 and day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates . Cell number and category of BALF samples were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice ( n = 3 per strain ) on day 1 and day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates . ( F ) Hematoxylin and eosin ( H & E ) - staining of lung sections were from EphA2 + / + and EphA2 − / − mice intranasally infected with reovirus ( 4 × 104 PFU per mouse ) on day 0 , day 1 and day 3 . Scale bars represent 200μm . ( G ) Immunoblot analysis of EphA2 and IL - 1β in lung homogenates were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice on day 1 and day 3 of intranasal infection with reovirus ( 4 × 104 PFU per mouse ) , β - actin served as a loading control throughout .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A-B) ELISA of IL-1β and IL-18 in BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.Cell number of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.Cell number and category of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.(F) Hematoxylin and eosin (H&E)-staining of lung sections were from EphA2+/+ and EphA2−/− mice intranasally infected with reovirus (4×104 PFU per mouse) on day 0, day 1 and day 3. Scale bars represent 200μm.(G) Immunoblot analysis of EphA2 and IL-1β in lung homogenates were from wild-type (WT) and EphA2-kockout (KO) mice on day 1 and day 3 of intranasal infection with reovirus (4×104 PFU per mouse), β-actin served as a loading control throughout.


Figure 4Quantitative RT - PCR analysis of mRNA encoding various cytokines ( IL - 4 , IL - 5 , IL - 13 in lung samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 4 per strain ) sensitized with ova ; results presented relative to β - actin . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates . Quantitative RT - PCR analysis of mRNA encoding various cytokines IL - 33 ) in lung samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 4 per strain ) sensitized with ova ; results presented relative to β - actin . Shown is mean ± SD . ( C ) ELISA analysis of IL - 33 in lung tissue homogenate samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates . ( D - G ) ELISA analysis of IL - 4 , IL - 13 , IL - 1β and IL - 18 in lung tissue homogenate samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates . ( H ) Cell number and category of BALF samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates . ( I ) H & E staining and PAS staining of lung sections from wild - type ( WT ) and EphA2 - knockout ( KO ) mice sensitized with ova . Scale bars represent 200μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Quantitative RT-PCR analysis of mRNA encoding various cytokines (IL-4, IL-5, IL-13 in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.Quantitative RT-PCR analysis of mRNA encoding various cytokines IL-33) in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. (C) ELISA analysis of IL-33 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.(D-G) ELISA analysis of IL-4, IL-13, IL-1β and IL-18 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.(H) Cell number and category of BALF samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.(I) H&E staining and PAS staining of lung sections from wild-type (WT) and EphA2-knockout (KO) mice sensitized with ova. Scale bars represent 200μm.


Figure 5 ( A ) Immunoblot analysis of proteins precipitated with anti - EphA2 or IgG ( control ) were from whole - cell lysates of AECs from wild - type mice stimulated with reovirus ( MOI = 5 ) for 3 h . Input , 10 % of the AECs lysate . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates . ( C ) The indicated plasmids of HA - tagged full - length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc - tagged NLRP3 , and then cell lysates were immunoprecipitated with anti - Myc antibody , followed by western blot analysis with anti - HA antibody and anti - Myc Antibody . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates . ( D ) The indicated plasmids of HA - tagged full - length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc - tagged EphA2 , and then cell lysates were immunoprecipitated with anti - Myc antibody , followed by western blot analysis with anti - HA antibody and anti - Myc antibody . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates . ( E ) Colocalization of endogenous EphA2 and NLRP3 in AEC cells . AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis . EphA2 was stained with rabbit anti - EphA2 ( 1 : 500 ) , followed by Alexa Fluor 594 goat anti - rabbit secondary antibody ( red ) , while NLRP3 was stained with mouse anti - NLRP3 ( 1 : 500 ) , followed by Alexa Fluor 488 goat anti - mouse secondary antibody ( green ) . DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) served as the nuclei marker ( blue ) . Scale bars represent 20μm . The Pearson ' s correlation coefficient and the Mander ' s overlap coefficient in the upper red box are 0 . 5 , and 0 . 95 respectively , and the lower red box 0 . 78 , and 0 . 94 respectively .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Immunoblot analysis of proteins precipitated with anti-EphA2 or IgG (control) were from whole-cell lysates of AECs from wild-type mice stimulated with reovirus (MOI=5) for 3 h. Input, 10% of the AECs lysate. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.(C) The indicated plasmids of HA-tagged full-length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged NLRP3, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc Antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.(D) The indicated plasmids of HA-tagged full-length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged EphA2, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.(E) Colocalization of endogenous EphA2 and NLRP3 in AEC cells. AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis. EphA2 was stained with rabbit anti-EphA2 (1:500), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (red), while NLRP3 was stained with mouse anti-NLRP3 (1:500), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker (blue). Scale bars represent 20μm. The Pearson's correlation coefficient and the Mander's overlap coefficient in the upper red box are 0.5, and 0.95 respectively, and the lower red box 0.78, and 0.94 respectively.


Figure 6 ( A ) The indicated plasmids of Myc - tagged NLRP3 and GFP - tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti - Myc antibody , followed by western blot analysis with the anti - p - Tyr antibody , anti - GFP and anti - Myc . ( B ) Immunoblot analysis of p - Tyr of NLRP3 precipitated with anti - NLRP3 antibody in AECs without ( Ctrl ) or with LPS stimulation or reovirus ( Reo ) infection . ( C ) The indicated plasmids of Myc - tagged wild - type NLRP3 or its mutants Y132F , Y164F , Y251F , Y570F and Y589F were respectively transfected into HEK293T cells along with GFP - tagged EphA2 , and then cell lysates were immunoprecipitated with the anti - Myc antibody , followed by western blot analysis with the anti - p - Tyr antibody , anti - HA antibody and anti - Myc antibody . ( D ) Immunoblot analysis of EphA2 , ASC , NLRP3 , caspase - 1 and IL - 1β in HEK293T cells were co - transfected with 200ng EphA2 , 40ng ASC , 40ng caspase - 1 and 200ng IL - 1β , vector or wild NLPR3 or Y132F as indicated . The position of protein markers ( shown in kDa ) is indicated on the right - hand side . ( E ) Densitometry analysis * P < 0 . 05 ( unpaired t test ) . NS means no statistical difference . Shown is mean ± SD . Data represent three independent biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) The indicated plasmids of Myc-tagged NLRP3 and GFP-tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-GFP and anti-Myc.(B) Immunoblot analysis of p-Tyr of NLRP3 precipitated with anti-NLRP3 antibody in AECs without (Ctrl) or with LPS stimulation or reovirus (Reo) infection.(C) The indicated plasmids of Myc-tagged wild-type NLRP3 or its mutants Y132F, Y164F, Y251F, Y570F and Y589F were respectively transfected into HEK293T cells along with GFP-tagged EphA2, and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-HA antibody and anti-Myc antibody.(D) Immunoblot analysis of EphA2, ASC, NLRP3, caspase-1 and IL-1β in HEK293T cells were co-transfected with 200ng EphA2, 40ng ASC, 40ng caspase-1 and 200ng IL-1β, vector or wild NLPR3 or Y132F as indicated. The position of protein markers (shown in kDa) is indicated on the right-hand side.(E) Densitometry analysis *P<0.05 (unpaired t test). NS means no statistical difference. Shown is mean ± SD. Data represent three independent biological replicates.


Figure 7 ( A - B ) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection ( MOI ) of 20 for 3h . AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control . ASC was stained with mouse anti - ASC ( 1 : 200 ) , followed by Alexa Fluor 488 goat anti - mouse secondary antibody ( green ) . DAPI served as the nuclei marker ( blue ) . Data information : Scale bars represent 50μm . Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates . ( C - D ) Confocal microscopy analysis of HEK293T cells transfected with flag - ASC and Myc - tagged vector or Myc - tagged NLRP3 or Myc - tagged NLRP3 Y132F . Cells were stained with anti - flag ( 1 : 200 ) , anti - Myc ( 1 : 200 ) , followed by Alexa Fluor 488 goat anti - rabbit secondary antibody ( green ) or Alexa Fluor 594 goat anti - mouse secondary antibody ( red ) . DAPI served as the nuclei marker ( blue ) . Data information : Scale bars represent 50μm . Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates .

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Figure 7(A-B) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection (MOI) of 20 for 3h. AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control. ASC was stained with mouse anti-ASC (1:200), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.(C-D) Confocal microscopy analysis of HEK293T cells transfected with flag-ASC and Myc-tagged vector or Myc-tagged NLRP3 or Myc-tagged NLRP3 Y132F. Cells were stained with anti-flag (1:200), anti-Myc (1:200), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody (green) or Alexa Fluor 594 goat anti-mouse secondary antibody (red). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


Figure 1A - B . SH - SY5Y cells and primary cortical neurons were treated with 1 μM α - syn fibrils ( A ) or monomers ( B ) for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 compared to PBS , unpaired t - test . C . Frontal cortices of A53T α - syn heterozygous transgenic mice and control mice at the age of 9 months ( old ) or 2 months ( young ) were lysed and Western blot was performed with the indicated antibodies ( n = 3 ) . * * * P < 0 . 001 compared to WT , unpaired t - test . FcγRIIB KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA . SHP - 1 KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA . SHP - 2 KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA . SH - SY5Y cells with 20 μM NSC87877 , 10 μM SSG and 1 μM PHPS - 1 were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-B. SH-SY5Y cells and primary cortical neurons were treated with 1 μM α-syn fibrils (A) or monomers (B) for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to PBS, unpaired t-test.C. Frontal cortices of A53T α-syn heterozygous transgenic mice and control mice at the age of 9 months (old) or 2 months (young) were lysed and Western blot was performed with the indicated antibodies (n=3). *** P < 0.001 compared to WT, unpaired t-test.FcγRIIB KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.SHP-1 KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.SHP-2 KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.SH-SY5Y cells with 20 μM NSC87877, 10 μM SSG and 1 μM PHPS-1 were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


Figure 2SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . The cells in the lower chamber were fixed and immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 against PBS , one - way ANOVA . NT , c - src KD # 1 and # 2 SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells for 12 h , and then fixed and immunostained with anti - EGFP antibody . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 compared to NT cells , one - way ANOVA , D . primary cortical neurons were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . The cells in the lower chamber were fixed and immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 against PBS , one - way ANOVA . , E . primary cortical neurons downregulating c - src were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells for 12 h , and then fixed and immunostained with anti - EGFP antibody . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 compared to NT cells one - way ANOVA unpaired t - testSH - SY5Y cells were transfected with plasmids of WT c - src , Y529F c - src ( kinase active ) , or K297M c - src ( kinase dead ) tagged with EGFP . After 1 day , the cells were cocultured with differentiated α - syn OE SH - SY5Y cells for 12 h and then fixed and immunostained with anti - α - syn antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * P < 0 . 05 compared to mock positive cells , one - way ANOVA . G . FcγRIIB KD SH - SY5Y cells were transfected with plasmids of WT c - src , Y529F c - src ( kinase active ) , or K297M c - src ( kinase dead ) tagged with EGFP . After 1 day , the cells were cocultured with differentiated α - syn OE SH - SY5Y cells for 12 h and then fixed and immunostained with anti - α - syn antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * P < 0 . 05 compared to mock positive cells , one - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.NT, c-src KD #1 and #2 SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells, one-way ANOVA, D. primary cortical neurons were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA., E. primary cortical neurons downregulating c-src were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells one-way ANOVA unpaired t-testSH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.G. FcγRIIB KD SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.


Figure 3B . A53T α - syn - EGFP and A53T α - syn - mCherry OE SH - SY5Y cells were cocultured in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 , respectively , with 50 μM RA for 5 days . C . NT or c - src KD # 1 or # 2 / A53T α - syn - EGFP and A53T α - syn - mCherry OE SH - SY5Y cells were cocultured with 50 μM RA for 5 days . The samples were observed under confocal microscopy , and the number of cells containing double fluorescence labeled puncta were analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to PBS or NT cells , one - way ANOVA .

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Figure 3B. A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1, respectively, with 50 μM RA for 5 days.C. NT or c-src KD #1 or #2 /A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured with 50 μM RA for 5 days. The samples were observed under confocal microscopy, and the number of cells containing double fluorescence labeled puncta were analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to PBS or NT cells, one-way ANOVA.


Figure 4A . Differentiated A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . B . Differentiated NT , c - src KD # 1 or c - src KD # 2 / A53T α - syn - EGFP OE SH - SY5Y cells were cultured 12 h . Levels of α - syn in culture media were then measured using ELISA . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 compared to control , one - way ANOVA . D . SH - SY5Y cells in the lower chamber were cocultured with differentiated NT , c - src KD # 1 or c - src KD # 2 / A53T α - syn - EGFP OE SH - SY5Y cells for 12 h . The cells in the lower chamber were immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to NT cells , one - way ANOVA . E . Differentiated A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 5 μM saracatinib ( sara ) or 10 μM SKI - 1 in the presence of DMSO or 50 μM bafilomycin A1 ( BafA ) for 12 h . Differentiated NT and c - src KD / A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 50 μM BafA for 12 h . Western blot was performed . Data are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA . G . NT and c - src KD primary neurons were incubated with 50 μM BafA for 12 h . Western blot was performed . Data are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA . H . SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h . The cells were immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * P < 0 . 01 , one - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h.B. Differentiated NT, c-src KD #1 or c-src KD#2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured 12 h. Levels of α-syn in culture media were then measured using ELISA. Values were derived from three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to control, one-way ANOVA.D. SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, c-src KD #1 or c-src KD #2/ A53T α-syn-EGFP OE SH-SY5Y cells for 12 h. The cells in the lower chamber were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to NT cells, one-way ANOVA.E. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 in the presence of DMSO or 50 μM bafilomycin A1 (BafA) for 12 h.Differentiated NT and c-src KD/A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.G. NT and c-src KD primary neurons were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.H. SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h. The cells were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). ** P < 0.01, one-way ANOVA.


Figure 5SH - SY5Y cells in the presence of 5 μM saracatinib , 10 μM SKI - 1 were incubated with 1 μM α - syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5 - LacCer and 2 . 5 μg / ml rhodamine - conjugated transferrin for 20 min . The cells were fixed and observed under confocal microscopy . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to control , one - way ANOVA . B . NT , c - src KD # 1 or c - src KD # 2 SH - SY5Y cells were incubated with 1 μM α - syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5 - LacCer and 2 . 5 μg / ml rhodamine - conjugated transferrin for 20 min . The cells were fixed and observed under confocal microscopy . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to control , one - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5SH-SY5Y cells in the presence of 5 μM saracatinib, 10 μM SKI-1 were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.B. NT, c-src KD #1 or c-src KD #2 SH-SY5Y cells were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.


Figure 6Two to three - month - old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α - syn fibrils . The mice were treated with saracatinib at 10 mg / kg / day by oral gavage for 4 weeks . B . Fixed brain tissues containing the frontal cortex and the striatum were lysed . Western blot was performed with indicated antibodies . Values were derived from three individual mice ( n = 3 ) . Two to three - month - old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α - syn fibrils . The mice were treated with saracatinib at 10 mg / kg / day by oral gavage for 4 weeks . C . Immunohistochemistry of pSer129 α - syn was performed . Arrow indicates pSer129 α - syn positive inclusion . Scale bar indicates 50 μm . D . Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions . Values were derived from eight individual mice ( n = 8 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS group , unpaired t - test . Data are expressed as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. B. Fixed brain tissues containing the frontal cortex and the striatum were lysed. Western blot was performed with indicated antibodies. Values were derived from three individual mice (n=3).Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. C. Immunohistochemistry of pSer129 α-syn was performed. Arrow indicates pSer129 α-syn positive inclusion. Scale bar indicates 50 μm. D. Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions. Values were derived from eight individual mice (n=8). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS group, unpaired t-test. Data are expressed as mean ± SEM.


Figure 7A . Immunohistochemistry of GFAP was performed . GFAP intensities in the cortex and the striatum were analyzed . Values were derived from three individual mice ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA . B . Immunohistochemistry of IBA - 1 and MHC Class II were performed . Arrow indicates MHC class II positive cells . Numbers of MCH class II positive cells in the cortex and the striatum were analyzed . Values were derived from three individual mice ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , one - way ANOVA .

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Figure 7A. Immunohistochemistry of GFAP was performed. GFAP intensities in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.B. Immunohistochemistry of IBA-1 and MHC Class II were performed. Arrow indicates MHC class II positive cells. Numbers of MCH class II positive cells in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). *** P < 0.001, ** P < 0.01, one-way ANOVA.


Figure 1A . RNA - seq of whole blood from symptomatic ( n = 26 ; median 8 days PIO ) and asymptomatic ( n = 30 ; median 3 days post - hospital admission ) COVID - 19 patients at the acute phase of infection ( SARS - CoV - 2 PCR - positive ) was performed . Only samples with RNA integrity number > 6 were sent for sequencing and included in the analysis . Volcano plot indicating DEGs between asymptomatic and symptomatic COVID - 19 patients , with thresholds of FDR < 0 . 05 and | FC | > 2 . Numbers of over - expressed and under - expressed genes are indicated .

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Figure 1A. RNA-seq of whole blood from symptomatic (n=26; median 8 days PIO) and asymptomatic (n=30; median 3 days post-hospital admission) COVID-19 patients at the acute phase of infection (SARS-CoV-2 PCR-positive) was performed. Only samples with RNA integrity number >6 were sent for sequencing and included in the analysis. Volcano plot indicating DEGs between asymptomatic and symptomatic COVID-19 patients, with thresholds of FDR < 0.05 and |FC| > 2. Numbers of over-expressed and under-expressed genes are indicated.


Figure 2B . Network analysis of significant immune mediators between symptomatic and asymptomatic COVID - 19 patients . Interactive relationships between the cytokines or chemokines were determined by STRING ( Search Tool for the Retrieval of Interacting Genes / Proteins ) analysis , with a confidence threshold of 0 . 5 .

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Figure 2B. Network analysis of significant immune mediators between symptomatic and asymptomatic COVID-19 patients. Interactive relationships between the cytokines or chemokines were determined by STRING (Search Tool for the Retrieval of Interacting Genes/ Proteins) analysis, with a confidence threshold of 0.5.


Figure 3C . SARS - CoV - 2 specific CD4 + non T follicular helper ( TFH ) cells from symptomatic ( n = 5 ) and asymptomatic COVID - 19 ( n = 5 ) patients were characterized with flow cytometry based on the expression of IL - 17a , IFN - γ , TNF - ⍺ , IL - 4 and IL - 10 upon SARS - CoV - 2 peptide stimulation . Representative plot overlay of unstimulated ( black ) on peptide stimulated ( red ) was performed on asymptomatic patient . Data are presented as Mean ± SD * * P < 0 . 01 ( Mann - Whitney U test ) .

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Figure 3C. SARS-CoV-2 specific CD4+ non T follicular helper (TFH) cells from symptomatic (n=5) and asymptomatic COVID-19 (n=5) patients were characterized with flow cytometry based on the expression of IL-17a, IFN-γ, TNF-⍺, IL-4 and IL-10 upon SARS-CoV-2 peptide stimulation. Representative plot overlay of unstimulated (black) on peptide stimulated (red) was performed on asymptomatic patient. Data are presented as Mean ± SD **P<0.01 (Mann-Whitney U test).


Figure 4A , B . ( A ) IgG and ( B ) IgM responses were analysed by screening plasma samples of asymptomatic ( n = 39 , median study day 29 ) and symptomatic ( n = 57 , median study day 31 ) COVID - 19 patients . Data are presented as Mean ± SD * * * P < 0 . 001 ( Mann - Whitney U test ) .

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Figure 4A, B. (A) IgG and (B) IgM responses were analysed by screening plasma samples of asymptomatic (n=39, median study day 29) and symptomatic (n=57, median study day 31) COVID-19 patients. Data are presented as Mean ± SD ***P<0.001 (Mann-Whitney U test).


Figure 5A . Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic ( n = 16 ) and symptomatic ( n = 52 ) COVID - 19 patients and healthy controls ( n = 17 ) . Data are presented as Mean ± SD * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) . B . Mass cytometry was performed on PBMCs obtained from acute asymptomatic ( n = 19 ) and acute symptomatic ( n = 37 ) COVID - 19 patients and healthy donors ( n = 10 ) . Monocytes were characterized based on their CD14 and CD16 expression . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) . C . Classical , intermediate and non - classical monocytes were further defined in acute asymptomatic ( n = 19 ) and acute symptomatic ( n = 37 ) COVID - 19 patients and healthy donors ( n = 10 ) with inflammatory marker CD169 . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) . D . Expression data of genes associated with inflammatory monocytes , activated neutrophils and pro - inflammatory cytokines were compared between acute asymptomatic ( n = 30 ) and asymptomatic ( n = 26 ) COVID - 19 patients and were represented by ratio with respect to expression in asymptomatic patients . Bar graphs show DEGs with FDR adjusted P value < 0 . 05 and fold change values are represented in the log2 scale . E . Pro - inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45 - plex microbead - based immunoassay . Immune mediator levels were compared between asymptomatic patients ( n = 48 ) and symptomatic patients stratified by COVID - 19 severity ( mild , n = 61 ; moderate , n = 43 ; severe , n = 68 ) . Immune mediator levels for healthy controls ( HC ) ( n = 23 ) are indicated by the black dotted line . Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification ( LOQ ) . Data are presented as Mean ± SD * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( One - way ANOVA with post hoc t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic (n=16) and symptomatic (n=52) COVID-19 patients and healthy controls (n=17). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).B. Mass cytometry was performed on PBMCs obtained from acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10). Monocytes were characterized based on their CD14 and CD16 expression. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison). C. Classical, intermediate and non-classical monocytes were further defined in acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10) with inflammatory marker CD169. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).D. Expression data of genes associated with inflammatory monocytes, activated neutrophils and pro-inflammatory cytokines were compared between acute asymptomatic (n=30) and asymptomatic (n=26) COVID-19 patients and were represented by ratio with respect to expression in asymptomatic patients. Bar graphs show DEGs with FDR adjusted P value < 0.05 and fold change values are represented in the log2 scale.E. Pro-inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45-plex microbead-based immunoassay. Immune mediator levels were compared between asymptomatic patients (n=48) and symptomatic patients stratified by COVID-19 severity (mild, n=61; moderate, n=43; severe, n=68). Immune mediator levels for healthy controls (HC) (n=23) are indicated by the black dotted line. Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification (LOQ). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA with post hoc t test).


Figure 6A . Growth factors in the plasma of asymptomatic ( n = 48 ) , symptomatic patients ( n = 173 ) and healthy controls ( n = 23 ) were analyzed by Luminex . A ratio of VEGF - A / VEGF - D was calculated and used to perform receiver operating characteristics analysis . The area under the curve was shown . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) . B . RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown . Genes with false discovery rate less than 0 . 05 ( * ) , 0 . 01 ( * * ) or 0 . 001 ( * * * ) are indicated . C . Cell counts of DCs , CD4 T cells , CD8 T cells and B cells that express specific chemokine markers between asymptomatic ( n = 19 ) , symptomatic patients ( n = 37 ) and healthy controls ( n = 10 ) are analyzed by CyTOF and shown . Data are presented as Mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Growth factors in the plasma of asymptomatic (n=48), symptomatic patients (n=173) and healthy controls (n=23) were analyzed by Luminex. A ratio of VEGF-A/VEGF-D was calculated and used to perform receiver operating characteristics analysis. The area under the curve was shown. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).B. RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown. Genes with false discovery rate less than 0.05 (*), 0.01 (**) or 0.001 (***) are indicated.C. Cell counts of DCs, CD4 T cells, CD8 T cells and B cells that express specific chemokine markers between asymptomatic (n=19), symptomatic patients (n=37) and healthy controls (n=10) are analyzed by CyTOF and shown. Data are presented as Mean ± SD. *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


Figure 1A . Pull - down assays of yTBP ( 61 - 240 ) with Kap95p , Kap114p and Kap121p . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap95p , Kap114p and Kap121p . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . B . Band intensities of Kap - βs from the SDS - PAGE gels in ( A ) were quantified and normalized by GST - yTBP intensity . Quantitative plots show percent Kap - βs binding on yTBP . C - E . ITC titration curves ( upper ) and binding isotherms ( lower ) of ( C ) Kap114p , ( D ) Kap95p , and ( E ) Kap121p with yTBP ( 61 - 240 ) . Salt concentration and Kd values are indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Pull-down assays of yTBP (61-240) with Kap95p, Kap114p and Kap121p. GST-fused yTBP (61-240) was incubated with recombinant Kap95p, Kap114p and Kap121p. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. B. Band intensities of Kap -βs from the SDS-PAGE gels in (A) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.C-E. ITC titration curves (upper) and binding isotherms (lower) of (C) Kap114p, (D) Kap95p, and (E) Kap121p with yTBP (61-240). Salt concentration and Kd values are indicated.


Figure 2A , B . Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC . SEC ( Superdex 200 ) elution profiles of the ( A ) Kap114p  RanQ69L and ( B ) Kap114p  yTBP complexes . Peak fractions were analyzed by SDS - PAGE and stained with Coomassie blue . The void volume ( Vo ) of the peak fraction and absorbance ( a . u . ) at 280 nm for each complex is indicated . SV - AUC analysis of ( C ) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards ( in brackets ) . SV - AUC ( D ) Kap114p and ( E ) the Kap114p  yTBP complex . The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards ( in brackets ) . F . SE - AUC analysis of the Kap114p  yTBP complex . The average molecular mass determined by the Sedfit program was ~ 133 . 5 kDa . The lower panel shows the residual difference ( standard deviation ) between experimental and fitted values . Plots of the distance distribution function P ( r ) for the ( G ) Kap114p  RanQ69L complexes derived from experimental SAXS data using the GNOM program . Rg - based dimensionless Kratky plot of the Kap114p  RanQ69L ( H ) complexes . Intersection of the two dotted gray lines indicates features of a compact protein . The ab initio envelopes determined by GASBOR for the ( I ) Kap114p  RanQ69L complexes . Models of the ( J ) Kap114p  RanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR . Plots of the distance distribution function P ( r ) for the ( K ) Kap114p  yTBP complexes derived from experimental SAXS data using the GNOM program . Rg - based dimensionless Kratky plot of the Kap114p  yTBP ( L ) complexes . Intersection of the two dotted gray lines indicates features of a compact protein .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, B. Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC. SEC (Superdex 200) elution profiles of the (A) Kap114pRanQ69L and (B) Kap114pyTBP complexes. Peak fractions were analyzed by SDS-PAGE and stained with Coomassie blue. The void volume (Vo) of the peak fraction and absorbance (a.u.) at 280 nm for each complex is indicated.SV-AUC analysis of (C) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).SV-AUC (D) Kap114p and (E) the Kap114pyTBP complex. The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).F. SE-AUC analysis of the Kap114pyTBP complex. The average molecular mass determined by the Sedfit program was ~133.5  kDa. The lower panel shows the residual difference (standard deviation) between experimental and fitted values.Plots of the distance distribution function P(r) for the (G) Kap114pRanQ69L complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pRanQ69L (H) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.The ab initio envelopes determined by GASBOR for the (I) Kap114pRanQ69L complexes. Models of the (J) Kap114pRanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR.Plots of the distance distribution function P(r) for the (K) Kap114pyTBP complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pyTBP (L) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.


Figure 3B . Pull - down assays of yTBP with Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . C . Band intensities of Kap - βs from the SDS - PAGE gels in ( B ) were quantified and normalized by GST - yTBP intensity . Quantitative plots show percent Kap - βs binding on yTBP . D , E . ITC titration curves ( upper ) and binding isotherms ( lower ) of ( D ) Kap114p ( Δ347 - 371 ) and ( E ) Kap114p ( Δ899 - 956 ) with yTBP ( 61 - 240 ) . Salt concentration and Kd values are indicated . F . Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type , KAP114 knockout strains , and KAP114 knockout strains rescued by KAP114 deletion mutants . G . His - tagged RanQ69L was incubated with recombinant Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . H . Band intensities of Kap - βs from the SDS - PAGE gels in ( G ) were quantified and normalized by His - Ran intensity . Quantitative plots show percent Kap - βs binding on His - Ran . I . GST pull - down assays of yTBP ( 61 - 240 ) with Kap114p or Kap114p ( Δ347 - 371 ) in the presence or absence of RanQ69L . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap114p and His - tagged RanQ69L at a molar ratio of 1 : 4 : 8 Kap114p : yTBP : RanQ69L . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . J . Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L . Band intensities of Kap114p and Kap114p ( Δ347 - 371 ) from the SDS - PAGE gels were used to determine the average fraction of bound protein .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Pull-down assays of yTBP with Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). GST-fused yTBP (61-240) was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. C. Band intensities of Kap-βs from the SDS-PAGE gels in (B) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.D, E. ITC titration curves (upper) and binding isotherms (lower) of (D) Kap114p (Δ347-371) and (E) Kap114p (Δ899-956) with yTBP (61-240). Salt concentration and Kd values are indicated.F. Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type, KAP114 knockout strains, and KAP114 knockout strains rescued by KAP114 deletion mutants.G. His-tagged RanQ69L was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. H. Band intensities of Kap-βs from the SDS-PAGE gels in (G) were quantified and normalized by His-Ran intensity. Quantitative plots show percent Kap-βs binding on His-Ran. I. GST pull-down assays of yTBP (61-240) with Kap114p or Kap114p (Δ347-371) in the presence or absence of RanQ69L. GST-fused yTBP (61-240) was incubated with recombinant Kap114p and His-tagged RanQ69L at a molar ratio of 1:4:8 Kap114p:yTBP:RanQ69L. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. J. Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L. Band intensities of Kap114p and Kap114p (Δ347-371) from the SDS-PAGE gels were used to determine the average fraction of bound protein.


Figure 4B . EMSA analysis of yTBP binding to DNA . We incubated 5 nM FAM - labeled oligonucleotide ( TGTATGTATATAAAAC ) with indicated concentrations of purified yTBP ( 0 . 125 to 8 μM ) and then analyzed binding by 6 % non - denaturing PAGE . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) . C . Indicated concentrations ( 0 . 25 to 16 μM ) of purified recombinant Kap114p were incubated with yTBP ( 4 μM ) and FAM - labeled DNA ( 5 nM ) , followed by native PAGE analysis . Kap114p ( 16 μM ) was incubated with DNA without yTBP as a control . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) . D . Super - shift EMSA analysis of TFIIA binding to yTBP  DNA complex . We incubated 5 nM FAM - labeled DNA and yTBP ( 0 . 5 μM ) with indicated concentrations of purified TFIIA ( 0 . 08 to 5 μM ) and then analyzed binding by 6 % non - denaturing PAGE . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) . E . Indicated concentrations ( 0 . 06 to 4 μM ) of purified Kap114p were added to the reaction containing 0 . 5 μM of TFIIA , yTBP and FAM - labeled DNA ( TGTATGTATATAAAAC ) , before conducting native PAGE analysis . TFIIA and Kap114p ( 4 μM ) were incubated with DNA without yTBP as a control . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) . Electrophoretic bands containing protein - DNA complex and free DNA are indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. EMSA analysis of yTBP binding to DNA. We incubated 5 nM FAM-labeled oligonucleotide (TGTATGTATATAAAAC) with indicated concentrations of purified yTBP (0.125 to 8 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).C. Indicated concentrations (0.25 to 16 μM) of purified recombinant Kap114p were incubated with yTBP (4 μM) and FAM-labeled DNA (5 nM), followed by native PAGE analysis. Kap114p (16 μM) was incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).D. Super-shift EMSA analysis of TFIIA binding to yTBPDNA complex. We incubated 5 nM FAM-labeled DNA and yTBP (0.5 μM) with indicated concentrations of purified TFIIA (0.08 to 5 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).E. Indicated concentrations (0.06 to 4 μM) of purified Kap114p were added to the reaction containing 0.5 μM of TFIIA, yTBP and FAM-labeled DNA (TGTATGTATATAAAAC), before conducting native PAGE analysis. TFIIA and Kap114p (4 μM) were incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). Electrophoretic bands containing protein-DNA complex and free DNA are indicated.