sentence: For the dephosphorylation reactions shown in Figure 2 , aliquots of NPr phosphorylation reactions were mixed with SixA , SixA ( H8A ) , or buffer alone and incubated at 30 ° C for an additional 20 min before analysis by native gel electrophoresis.To follow NPr-P dephosphoryation over time in Figure 3 , NPr-P was purified by processing the phosphorylation reaction through a centrifugal filter unit ( 30 kDa cutoff ; Amicon UFC503008 , Millipore-Sigma ) to eliminate EI , concentrating the flow-through 2.5-fold with a centrifugal filter unit ( 3 kDa cutoff ; Amicon UFC503008 , Millipore-Sigma ) , and then spinning the ultrafiltrate through a desalting spin column ( Zeba 89882 , Thermo Scientific ) to reduce phosphoenolpyruvate levels .