Western blotting

Total protein was isolated as follows. First, the neurons were lysed using RIPA buffer containing NaF and protease inhibitors for 30 min on ice. Substantia nigra tissue obtained from mice was homogenized in ice-cold RIPA buffer containing NaF and protease inhibitors. The lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatant fractions were collected. The supernatant fractions were re-suspended in 1× SDS sample buffer and boiled at 98 °C for 5 min.

Cell fractionation was conducted as follows (Protein Assay Kit, Beyotime Biotechnology): cells were collected in PBS and washed twice with cold PBS. The cell pellet was re-suspended in hypotonic buffer and incubated on ice for 15 min. Next, buffer was added to the cell suspension, and the sample was vortexed for 10 s. The homogenate was centrifuged for 5 min at 12,000 × g and 4 °C to obtain the cytoplasmic (supernatant) and nuclear (pellet) fractions. The nuclear pellet was re-suspended in complete cell extraction buffer and incubated on ice for 30 min, with shaking at 5-min intervals. The nuclear lysate was centrifuged at 12,000 × g and 4 °C for 15 min to obtain the nuclear fraction (supernatant). Then, 60–100 μg of protein was used for western blotting. Samples were separated on 10% SDS-PAGE gels and transferred to a PVDF membrane. The membranes were sequentially incubated with primary and secondary antibodies. Proteins were detected using horseradish peroxidase-coupled goat anti-mouse IgG or anti-rabbit IgG antibodies and an ECL western blotting detection system.

Soluble and insoluble proteins were extracted as follows. Substantia nigra tissues obtained from mice were homogenized in an ice-cold cocktail of 1% Tx-100 in Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.4) containing protease and phosphatase protease inhibitors. Neurons were added to this cocktail at 4 °C. Lysates were centrifuged at 100,000 × g for 30 min, and the supernatant was harvested as a TX-soluble fraction. The TX-insoluble proteins were extracted using 2% (w/v) SDS/TBS and finally reconstituted in an equal volume of SDS/TBS buffer. Proteins were separated using SDS-PAGE. We confirmed all blots derive from the same experiment and were processed in parallel.