Ecl system

The total proteins were extracted from cerebral cortex by 1 × RIPA lysis buffer (Meilunbio, Dalian, China) containing protease inhibitors (Meilunbio, Dalian, China) and phosphatase inhibitors (Meilunbio, Dalian, China). A total of 25 μg proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. Then the PVDF membrane was in blocked with 5% skim milk powder solution in PBS-Tween 20 (PBST) for 60 min and incubated with primary antibodies overnight at 4°C. After that, the membrane was incubated with secondary antibodies for 40 min at room temperature. The blots were detected using ECL system (Vazyme Biotech, Nanjing, China) and captured by a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). The primary antibodies were as follows: KCNJ3 (Affinity, AF9101), ADCY5 (Bioss, bs-3922R), FAM213B (Abcam, Ab180932), and COX2 (Affinity, AF7003).

Tissues were homogenized in RIPA buffer (6.5 mM Tris, pH 7.4, 15 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.25% sodium deoxycholate, 1% NP-40). The homogenate was cleared by centrifugation at 4°C for 20 min at 20,627
g, and the supernatant containing proteins were collected. Bicinchoninic Acid reagents (Thermo Fisher Scientific) were used to determine the protein concentration. Equal amounts of proteins were resolved by 10% SDS-PAGE, followed by transfer onto PVDF membranes. The membranes were blocked with 5% BSA in Tris-buffered saline containing 0.2% Tween-20 (TBS-T), and then incubated with primary antibodies at 4°C overnight. The blots were finally immunoreacted with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using an enhanced chemoluminescence (ECL) system (Vazyme, Nanjing, China). The primary antibodies include anti-HSC70, anti-caspase-1 p10 and anti-IL-1β antibodies were from Santa Cruz Biotechnology; anti-SREBP2, anti-NLRP3 and anti-LXRα antibodies were from Abcam.