Figure 1

(A) α-syn binding assay. Step 1. Synaptosomes are prepared from α-syn^-/- mice and α-syn (human Wt and PD-linked A30P and A53T forms) is expressed and purified from E. coli. Step 2. Synaptic membranes (α-syn acceptor fraction) are prepared from intact synaptosomes using hypotonic buffer and incubated with purified α-syn (donor fraction) in presence or absence of α-syn^-/- (KO) cytosol. Step 3. Membrane and cytosol fractions are separated by centrifugation and the membrane proteins are analysed by western blotting. (B) Using the binding assay, KO synaptic membranes were incubated, for 10 min at 37°C, with 3 μg of Wt, A30P or A53T purified α-syn in absence or presence of 1.5 mg/ml of KO cytosol. As shown on this graph, A30P purified α-syn has a lower binding compared to Wt and A53T α-syn in absence (One-Way ANOVA, p < 0.0001, n = 4; Bonferroni's multiple comparison test) or presence (One-Way ANOVA, p < 0.0001, n = 4; Bonferroni's multiple comparison test) of KO cytosol. (C) KO synaptic membranes were incubated, for 10 min at 37°C, with 0.1, 0.6 and 3 μg of Wt, A30P or A53T purified α-syn in absence or presence of 1.5 mg/ml of KO cytosol. Results are normalized to the maximal binding observed for each respective α-syn. These data show that the cytosol has a significant effect by increasing the binding of all types of α-syn (One-Way ANOVA: Wt: p < 0.0001, n = 4; A30P: p < 0.0001, n = 4; A53T p < 0.001, n = 4).