Ab212184

Flash frozen cortex was homogenised in 1% Triton‐X100 with Complete protease inhibitors. Protein concentration was quantified using a BCA assay. Homogenate containing 1 mg protein was ultracentrifuged at 120,000 g (Beckman Optima Max‐XP) for 1 h at 4°C. The pellet was dispersed in 1% Triton‐X100 using a needle to produce a homogenate containing a Triton‐insoluble fraction. From this homogenate, a 5% input was removed for subsequent immunoblotting. The remaining triton‐insoluble fraction was incubated overnight at 4°C with the IP antibody against α‐synuclein (1:100, Abcam ab212184). The mixture was then incubated with 25 μl Protein G Dynabeads according to the manufacturer's instruction. The protein of interest was eluted using NuPAGE LDS sample buffer and separated by electrophoresis on a 4%–12% polyacrylamide gel. Immunoblotting was performed using 5% milk blocking with overnight incubation at 4°C with an α‐synuclein antibody (1:1000, BD 610787). The membrane was incubated with a horseradish‐peroxidase‐conjugated anti‐mouse antibody (1:20,000, Sigma A9917) for 1 h, and visualised with chemiluminescence.

SH-SY5Y cells and the substantia nigra from mice brains treated with or without reserpine were analyzed. Western blotting was conducted as reported previously. RIPA lysates containing protease and phosphatase inhibitors were used to obtain the protein samples (Li Y. et al., 2019b (link)). Protein concentrations were determined using the BCA quantification method (Pierce, Rockford, IL). The same quantity of protein samples (30 μg) was added per well in the 10% and 15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoresis proteins were transferred to the 0.45 μm PVDF membranes. The blots were blocked in 5% milk for 1 h. Blots were incubated overnight at 4°C with primary antibodies against alpha-synuclein (Abcam, ab212184), LC3 (Abcam, ab51520), p62 (CST, 8025), DNMT3b (Abcam, ab119282) and GAPDH (Mesgen biotechnology, MAN1002). On the next day, the blots were incubated with the secondary antibody, (goat anti-rabbit IgG (H + L) HRP (Mesgen, MAN4001), or goat anti-mouse IgG (H + L) HRP (Mesgen, MAN4002)) for 1 h at room temperature. The ECL detection solution was prepared as follows: A: B = 1:1, mixed homogeneously. Blots were imaged for detecting luminescence. Finally, the grayscale values were analyzed using Fiji Image J software.