Anti gapdh

The substantia nigra tissue was cut it into small pieces, and then 400 µL of single detergent lysis solution (including PMSF) was added. The solution was lysed for 30 min and centrifuged at 12,000 rpm at 4 °C for 5 min, and the supernatant was then collected. The concentration of the protein to be tested was measured and recorded and SDS–PAGE electrophoresis (voltage 40 V) was performed. The proteins were transferred to a membrane, which was stained with 1× Ponceau staining solution for 5 min, moved to 5% skim milk, sealed for 2 h, incubated with the primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; Cell Signaling Technology, Danvers, MA) for 2 h at room temperature, and finally subjected to chemiluminescence, development, and fixation. Following primary antibodies were used: anti-TH (1:1000; Sigma, T2928), anti-NLRP3 (1: 1000, BioVision, A1767-100), anti-IL-1β (1:1000; Sigma, I-3767); anti-Capase-1 (1:1000; Sino Biological, 90,011-MM02) anti-GAPDH (1:1000, Sino Biological, MB10094-T52).

Cells were lysed with RIPA buffer (Sangon), and proteins were quantified using a BSA Kit (Beyotime, Shanghai, China). Tumor proteins were extracted by a One-Step Animal Tissue Active Protein Extraction kit (Sangon). Protein (20 μg) was loaded and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Fdbio Science, Hangzhou, China) at 80 V for 30 min and then 120 V for 60 min followed by transfer to polyvinylidene fluoride (PVDF) membranes (Millipore) at 300 mA for 90 min. After blocking with 5% fat-free milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-Siglec-15 (1 : 1000, Santa Cruz), anti-GAPDH (1 : 5000, Sino Biological), anti-AGO2 (1 : 20, Abcam), anti-C-caspase3 (1 : 1000, Abcam), anti-caspase3 (1 : 1000, Abcam), and anti-Bcl-2 (1 : 1000, Abcam). After incubation with HRP-conjugated antibodies (1 : 5000, Sino Biological) for 2 h at room temperature, an ECL kit (Fdbio Science) was used for visualization of protein bands.

The anti-PD-L1 antibody, anti-KDM1A antibody, and anti-MEF2D antibody were purchased from Abcam. Anti-GAPDH, anti-HA, and anti-Flag were purchased from Sino Biological. Anti-K267me was generated from Abmart by immunization with 263-APSR(meK)PDLR-271 [22 (link)]. ORY-1001 and rotenone were purchased from Sigma. All primers and sgRNAs are shown in Table S1.