SDS-PAGE and Western Blot Analysis

Protein samples were treated for 30 min at 50°C in a sample-treating solution containing 2% SDS and 5% β-mercaptoethanol. The samples were then loaded onto a 12% or 15% SDS-polyacrylamide gel (7.0×8.3 cm ×0.75 mm) and electrophoresed using Mini-Protean Tetra system (Bio-Rad) at 15 mA (current constant). After electrophoresis, proteins separated on the gel were transferred onto a methanol-activated PVDF membrane (Immobilon-P, pore size 0.45 µm, Millipore) or a nitrocellulose membrane (Protran BA85, pore size 0.45 µm, Whatman) for 2 h using TE22 Mighty Small Transfer system (Hoefer Scientific) at 100 V (voltage constant). The membrane was then treated with or without phosphate-buffered saline (PBS) containing 0.4% PFA for 30 min at room temperature, followed by blocking for 1 h with 5% skim milk (Carnation) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). The membrane was then incubated for 1 h with a primary antibody in TBS-T containing 1% skim milk. As primary antibody, mouse monoclonal anti-α-syn antibodies 4D6 and LB509 (Santa Cruz Biotechnology) and rabbit monoclonal anti-phospho α-syn antibody EP1536Y (Epitomics, Burlingame, CA) were used at a dilution 1∶1,000, and rabbit polyclonal anti-actin antibody (Sigma) was also used at a dilution 1∶5,000. After washing with TBS-T containing 1% skim milk for 5 min three times, the membrane was incubated for 1 h with a secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody (Santa Cruz Biotechnology), in TBS-T containing 1% skim milk. After washing with TBS-T for 10 min three times, protein bands on the membrane were detected by chemiluminescence method using ECL-Plus immunoblotting detection system (GE Healthcare).