Anti α syn

α-syn, p53 and actin expressions were analyzed in various cell lines and mouse brain homogenates (50-200 μg of proteins) then loaded on 12-16 % sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and wet-transferred to Hybond-C (Amersham Life Science) membranes. Transferred proteins were immunoblotted using anti-α-syn (#2642, Cell Signaling), anti-flag (clone M2, F3165, Sigma), anti-p53 (CM1, provided by J.C. Bourdon) and anti-actin (clone AC-74, A5316, Sigma) antibodies. Immunological complexes were revealed with either anti-rabbit or anti-mouse IgG-coupled peroxidase antibodies (Jackson ImmunoResearch) by the electrochemiluminescence detection method (Roche Diagnostics S.A.S). Chemiluminescence was recorded using a luminescence image analyzer LAS-4000 (Raytest, Fuji) and quantifications of non-saturated images were performed with the FUJI Film Multi Gauge image analyzer software.

All samples were prepared using phosphate-buffered saline (PBS) supplemented with 10% goat serum and 0.1% Triton X-100. Next, the samples were incubated with blocking buffer (5% BSA in TBST, 1 TBS + 0.1% TWEEN 20 buffer) for 45 min at room temperature and then with anti-SUMO-1 (#4930, 1:100 dilution, Cell Signaling Technology, Boston, MA, USA) and anti-α-syn (#2628, 1:100 dilution, Cell Signaling Technology) primary antibodies, at 4°C overnight. After washed with PBS, the samples were incubated with secondary antibody [goat anti-rabbit IgG/fluorescein isothiocyanate (FITC) antibody (bs-0295G-FITC, 1:100 dilution, Bioss, Beijing, China)] for 1 h at room temperature. VECTASHIELD Antifade Mounting Medium with DAPI (H-1200, VECTOR, Burlingame, CA, USA) was used for nuclear labeling. Photomicrographs were captured using a fluorescence microscopy (A1+/A1R+, Nikon, Tokyo, Japan). All digital images were processed using the same settings to improve the contrast.

Total protein was extracted from cell pellets using RIPA Lysis buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% DOC, 1% NP-40, 0.1% SDS, and proteinase inhibitor cocktail), measured with the DC Protein Assay (Bio-Rad) and gels were blotted onto nitrocellulose membranes. Primary antibodies used overnight at 4 °C for immunoblotting were as follows: anti- α-Syn (1:1000 #2647 for cells; 1:600 #2642 for flies; Cell Signaling), anti-Cleaved Caspase-3 (1:1000 #9662; Cell Signaling), and anti-β-Actin (1:1000000 #A5316; Sigma). Secondary antibodies used for 1 h at room temperature for immunoblotting were as follows: anti-Rabbit IgG, HRP-linked antibody (1:2500; Cell Signaling) and anti-Mouse IgG, HRP-linked antibody (1:2000; Cell Signaling). All western blots presented in the same figure derived from the same experiment and were processed in parallel. In general, blots for replication experiments were processed under comparable conditions.