Western Blot Analysis of α-Synuclein

Gel electrophoresis was performed using 4%–12% or 12% Bolt Bis–Tris Plus gels (Thermo Scientific). Proteins were transferred to 0.45-μm polyvinylidene fluoride membranes for 60 min at 35 V. Proteins were crosslinked to the membrane via 0.4% (v/v) paraformaldehyde incubation in PBS for 30 min at room temperature, with rocking. The membranes were blocked for 60 min at room temperature in blocking buffer (5% [w/v] skim milk in 1× TBST (TBS and 0.05% [v/v] Tween-20)) and then incubated overnight at 4 °C with primary antibody directed against amino acids 15–123 of the α-synuclein protein (1:10,000 dilution, ref: 610786, BD Biosciences, Franklin Lakes, NJ) [8 (link), 24 (link)], diluted in the blocking buffer. The membranes were then washed three times with TBST and then incubated, for 60 min at room temperature, with horseradish peroxidase-conjugated secondary antibodies (ref: 172-1011, Bio-Rad, Hercules, CA) diluted 1:10,000 in the blocking buffer. Following another three washes with TBST, immunoblots were developed using Western Lightning enhanced chemiluminescence Pro (PerkinElmer, Waltham, MA) and imaged using X-ray film or the LiCor Odyssey Fc system.