Ab263899

Cells were washed with cold phosphate buffer saline (PBS) twice and lysed with RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (Thermo Scientific™ Halt). After centrifugation of cell lysates, proteins in supernatants were separated by SDS‐PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, blocked in 5% bovine serum albumin (BSA), and incubated with anti‐slc44a1 polyclonal antibody (1:1000, Proteintech), anti‐Caspase‐1 (1:1000, ab179515), anti‐NLRP3 (1:1000, ab263899), anti‐IL‐1β (1:1000, ab283818) overnight at 4°C. The secondary antibody (1:3000, Beyotime) was added to incubate with protein samples for 1 h, and detection was performed by the ECL chemiluminescence system (Bio‐Rad, Hercules).

Protein samples from the brain tissues were lysed in radioimmunoprecipitation assay buffer (Beyotime, Nantong, China) with 1 mM protease inhibitor (Boster, Wuhan, China). Protein concentrations were determined using a BCA protein assay kit (Beyotime, Nantong, China) was used to quantify Protein concentrations. Western blotting was conducted as previously reported [52 (link)]. Membranes were blocked with 8% non-fat dried milk at room temperature for 1 h, then incubated with primary antibodies GSDMDC1 (1:200, Santa Cruz, sc-393656), α7nAChR (1:100, Santa Cruz, sc-58607), ASC (1:200, Santa Cruz, sc-514414), NLRP3 (1:1000, Abcam, ab263899), caspase1 (1:1000, Proteintech, 22915-1-AP), GAPDH (1:5000, Proteintech, 10494-1-AP), and β-tubulin (1:5000, Proteintech, 10094-1-AP) overnight at 4 °C. After incubating the membranes with corresponding secondary antibodies, images were captured and quantified using the Fusion FX5 analysis system and analyzed using the Quantity One software.