Effect of cytosol on binding α-syn. (A) Recombinant α-syn (Wt, A30P and A53T) were incubated in presence of different concentrations of KO cytosol (0.5, 1.5, and 3 mg/ml), for 10 min at 37°C. Compared to the control condition (without cytosol), all cytosol concentrations had a significant effect on Wt and A30P α-syn binding, but only the highest concentration of cytosol had a significant effect on A53T α-syn binding (One way ANOVA test, p < 0.0001, Bonferroni's multiple comparison post-test). (B) KO synaptic membranes and α-syn were pre-incubated for 15 minutes at room temperature with KO cytosol. Membranes were then centrifuged at 14000 × g and washed with KOAc buffer to remove unbound factors. Binding of purified α-syn to KO membranes in the absence of cytosol (ctrl) was compared to its binding to cytosol-treated membranes without added cytosol (memb), and to cytosol-treated α-syn incubated with KO membranes (α-syn). No significant difference was observed between the two pre-incubated condition (Student T-test, p > 0.05). (C) KO cytosol was pre-incubated with trypsin or proteinase K for 15 min at 37°C. Enzymes were then respectively inactivated with trypsin inhibitor and PMSF for 10 min at room temperature. Compared to the cytosol condition (cyt) which, as a control, was incubated with the enzyme pre-inactivated by the inhibitor, only A30P α-syn binding was significantly affected by the cytosolic protein digestion (Student T-test, p < 0.0001), whereas no significant differences were observed for Wt and A53T proteins (Student T-test, p > 0.05).