Ab241996

Total protein was isolated from the BV2 cells using 100 μL precooled RIPA lysis buffer (Sigma‐Aldrich) with 1 μL protease inhibitor (ApexBio Technology) and centrifuged at 14,000 rpm at 4°C for 10 min. A BCA protein assay kit (Yeasen) was prepared for assessment of protein concentration. Protein (20 μg) was boiled in loading buffer, resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% nonfat milk, the membranes were incubated overnight with primary antibodies against Kir6.1 (ab241996, 1:1000; Abcam), SUR2A (Bio Excellence International Tech), Kir6.2 (ab79171, 1:1000; Abcam), SUR1 (ab217633, 1:1000; Abcam), and ACTIN (ab8227, 1:2000; Abcam) at 4°C. Then the membranes were incubated with secondary antibodies for 1 h at room temperature. Proteins were visualized by the enhanced chemiluminescence reagent (Yeasen). The immunoblot images were evaluated using ImageJ software.

Cell lysates and tissues were homogenized in RIPA lysis buffer (Beyotime Biotechnology, China) and protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). A 25-μg protein aliquot of each sample was separated and then electrophoretically transferred onto PVDF membranes (IPVH00010, Millipore, USA). Immuno-reactive bands were analyzed with ImageQuant™ LAS 4000 imaging system (GE Healthcare, Pittsburgh, PA, USA). Protein levels were determined by normalizing to the level of β-actin. The following primary and secondary antibodies were used: anti-NLRP3 (AG-20B-0014-C100, Adipogen), anti-caspase-1 (AG-20B-0042, Adipogen), anti-IL-1β (ab10626, Abcam), anti-GSDMD (ab219800, Abcam), anti-Kir6.1 (ab241996, Abcam), and anti-β-actin (BM0627, Boster, Pleasanton, CA, USA), Anti-mouse IgG, HRP-linked Antibody (7076, Cell Signaling Technology), Anti-rabbit IgG, HRP-linked Antibody (7074, Cell Signaling Technology).

The total cell lysates were prepared from WT and CKO astrocytes and then incubated with anti-Kir6.1 (ab241996, Abcam, 1:500 dilution), anti-NLRP3 (AG-20B-0014-C100, Adipogen, 1:800 dilution) or anti-ASC (sc-271054, Santa Cruz Biotechnology, 1:1000 dilution) antibodies at 4 °C overnight, followed by an incubation with 20 μl protein A/G plus agarose (sc-2003, Santa Cruz Biotechnology, USA) for 4 h at 4 °C. After washing the beads, the bound proteins were eluted and analyzed by immunoblotting.