Anti α syn c20

Fifty μg of sucrose gradient purified fractions (B, C, and D), diluted in PBS, were sonicated for 5 min in a water-bath sonicator, and run on a 13% SDS-PAGE Tris-glycine gel. Proteins were subsequently transferred onto nitrocellulose membranes (Amersham, 106000001) and analyzed by immunoblotting. For dot blot analysis, 20 μg of pre-sonicated fractions C were loaded on a multi-well device, and proteins were bound to a nitrocellulose membrane (Amersham, 10600001), under vacuum for 30 min at RT. Membranes were blocked in blocking buffer (5 % non-fat milk, 0.05% Tween-20 in TBS), for 1 h at RT and probed overnight at 4^oC with the following primary antibodies: anti-α-Syn C20 (Santa Cruz, 1:1000), anti-α-syn Syn1 (BD biosciences, 1:1,000), anti-pS129 α-Syn (Abcam, 1:1,000), anti-Flotillin-1 (Santa Cruz, 1:1,000), and recombinant anti-α-Syn aggregate antibody (conformation-specific MJFR-14-6-4-2, Abcam, 1:50,000). Following 10 min washes with 0.05% Tween-20 in TBS (TBS-T) thrice, secondary antibodies (Merck-Millipore) diluted in blocking buffer were added for 2 h at RT. Membranes were rinsed again as above and Clarity Western ECL Substrate (Bio-Rad) was used for the detection of the proteins bound to nitrocellulose membranes (Supplementary Table 6).

Cells were washed twice with PBS 1×, fixed in 4% paraformaldehyde for 20 min, washed twice with PBS 1× and treated with 0.1 M glycine for 4 min in PBS 1×, washed twice and permeabilized with 0.1% Triton X-100 in PBS 1× for 4 min. Cells were then incubated in blocking solution (0.2% BSA, 1% NGS, 0.1% Triton X-100 in PBS 1×), followed by incubation with primary antibodies diluted in blocking solution for 2:30 h at room temperature. After two washes in PBS 1×, cells were incubated with labelled secondary antibodies and 1 μg/ml DAPI (for nuclear staining) for 60 min. Cells were washed twice in PBS 1× and once in Milli-Q water and mounted with Vectashield mounting medium (Vector Lab, H-1000). The following antibodies were used: anti-FLAG 1:100 (Sigma–Aldrich, F7425), anti-MYC 1:250 (Cell Signaling, 2276), anti-α-syn (C-20) 1:200 (Santa Cruz Biotechnology, sc-7011-R), anti-α-syn (SYN-1) 1:200 (BD Transduction Laboratories, 610787) and anti-α-syn(phosphoS129) 1:200 (Abcam, ab59264). For detection, Alexa Fluor-488, −594 or −647 (Life Technologies) antibodies were used. Image acquisition was performed using C1 Nikon confocal microscope (60x oil, NA 1.49, 7× zoom-in). In the present study, data were collected from 6 independent experiments.