Syn211

Hemibrains were homogenised in RIPA buffer (SDS-PAGE) or non-denaturing lysis buffer (native PAGE), as previously described [34 (link)]. For immunoblot analysis, 10 μg of total protein per lane was loaded on 4–15% gels (Bio-Rad) and blotted onto polyvinylidene difluoride membranes (Bio-Rad). To determine the effects of vaccination on the levels of alpha-synuclein, MHCII and Fc-gamma receptor, blotted samples from immunised SNCA-OVX mice were probed with the rabbit MJFR1 antibody against human a-syn (Abcam, 1:1000), MHCII (eBioscience, 1:500) and Fc-gamma receptor CD16/32 (Abcam, 1:500). Overnight incubation at 4°C was followed by incubation in goat anti-rabbit secondary HRP antibody (Bio-Rad, 1:5000) for 1–2 h at room temperature, and visualisation with enhanced chemiluminescence (Millipore). Beta-actin was used as a loading control. To examine which species of a-syn are recognised by antibodies produced after immunisation, 0.1 μg of recombinant a-syn and a-syn oligomers were loaded on 4–15% SDS-PAGE gels and analysed by immunoblot using vaccination-elicited antibodies as primary antibody (1:500) and goat anti-mouse HRP antibody (Bio-Rad, 1:5000) as secondary antibody. The monoclonal antibody SYN211 (Abcam, 1:5000) served as positive control.

Cell lines or primary neuronal cultures were fixed in cold 2% paraformaldehyde for 20 min, washed in PBS, and permeabilized with 0.5% saponin in blocking solution (1% BSA in PBS (w/v)) for 30 min at room temperature. Cells were incubated with anti-α-Syn antibodies, Syn211 (at 1:750 dilution, overnight at 4 °C); MJFR-1 (1:2000), LB509 (ab27766; 1:250), or anti-α-Syn ab21976 (1:330) from Abcam, Zotal, Tel Aviv, Israel. The immunoreactive pattern of these anti-α-Syn antibodies in SK-Mel2 cells is shown in Fig. S1 (P-AP2M1-T156 (D4F3; Cell Signaling Technology; 1:300). The following antibodies were from Echelon Biosciences (Salt Lake City, UT, USA): anti-PI(3,4)P₂ (Z-P034b; 1:300) or anti-PI(4,5)P₂ (Z-P045 1:200; Z-A045 1:800) for 2 h at room temperature. Cells were then washed (PBS; 10 min ×3) and incubated with a host-suitable secondary ab, washed again, and mounted in Vectashield mounting medium (Vector Laboratory, Burlingame, CA USA).