Ab75487

The total protein of different treated cells or tissues were extracted by RIPA lysis buffer (P0013B, Beyotime Institute of Biotechnology) and centrifugation. After the concentration measurements by BCA assay kit (Shenergy Biocolor Bioscience and Technology Company, Shanghai, China), equal amount of protein was denatured and separated by 12% SDS-PAGE electrophoresis, then transferred to polyvinylidene fluoride membranes (PVDF; Immobilon-P, Cat. No. IPVH00010). The membranes were blocked in TBS containing 0.05% tween20 (TBST) with 5% BSA and incubated with related primary antibodies in TBST with 3% BSA overnight. Then, they were incubated in second antibodies for 1 h, detected by use of the ECL kit (Millipore, Hercules, CA, USA) and analyzed by ImageJ software. The primary antibodies were used as follows: anti-PINK1 antibody (ab75487, Abcam), anti-p53 antibody (sc1312, Santa Cruz), anti-LC3B antibody (3868s, CST) and anti-cleaved caspase 3 antibody (9664s, CST).

Ipsilateral basal cortical samples facing the blood clots were extracted. Western blot was performed as described previously^{37 (link)}. Briefly, cortical samples were homogenized, and centrifuged (1000 × g, 10 min, 4 °C). The supernatant was further centrifuged, and then, the protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). An equal amount of protein (50 μg) was suspended in loading buffer, then denatured at 95 °C for 5 min, and loaded on an SDS-PAGE gel. After electrophoresis and transfer onto polyvinylidene fluoride membranes, the membranes were blocked with non-fat dry milk buffer for 2 h and then incubated overnight at 4 °C with primary antibodies for LC3 (2775, 1:1000, Cell Signaling), Atg5 (ab54033, 1:500, Abcam), Parkin (ab15954, 1:1000, Abcam), PINK1 (ab75487, 1:500, Abcam), NLRP3 (ab98151, 1:800, Abcam), ASC (ab155970, 1:1000, Abcam), caspase-1 (ab1872, 1:1000, Abcam), IL-1β (SC-23460, 1:500; Santa Cruz), IL-18 (ab71495, 1:1000; Abcam). The membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein band densities were detected by X-ray film and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).