α-Syn binding to synaptosomal lipid rafts. Using a 42-30-5% discontinuous sucrose gradient, we analysed the proportion of α-syn that co-localised with flotilin-1, a lipid-raft marker. (A) Less then 5% of α-syn co-eluted with flotillin-1 after binding (in vitro) to α-syn KO synaptic membranes, in absence or presence of KO cytosol, and proportionally, no significant differences were observed between Wt and PD-linked mutants (Student's T-test, p > 0.05). (B) The proportion of α-syn that co-localised with flotillin-1 in vivo, using intact synaptosomes from transgenic mice expressing the human α-syn (Wt, A30P or A53T). As observed in vitro, only a small proportion of α-syn co-eluted with lipid rafts and no significant differences were observed between Wt and PD-linked mutations (Student T-test, p > 0.05). (C) A30P α-syn was subjected to paraformaldehyde-induced cross-linking to potential interacting proteins in synaptic membranes after 1, 2, 3, 5 and 10 minutes of incubation with synaptic membranes. A significant increase of bound α-syn after 2, 3 and 5 minutes was observed compared to the control condition (without cross-linking) (One-Way ANOVA p < 0.001, Bonferroni's multiple comparison test). (D) The proportion of α-syn present in the lipid-raft fraction after cross-linking did not show any significant differences compared to incubations without cross-linking (Student t-test: 1 min: p > 0.05; 3 min: p > 0.05). (E) A significant increase of bound Wt and A53T α-syn after paraformaldehyde-induced cross-linking was observed compared to the control condition without cross-linking (One-Way ANOVA, *p < 0.05, **p < 0.01,***p < 0.001, Bonferroni's multiple comparison test).