Rabbit anti asc

Hippocampal neurons and brain tissue samples were collected for Western blot analysis. Protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis gels and electrotransferred onto 0.22-μm polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) at RT for 1 h and then incubated with mouse anti-GPR120 (1:500; Santa Cruz, USA), mouse anti-Caspase-1 (1:500; Santa Cruz, USA), mouse anti-IL-1β (1:1000; CST, UK), mouse anti-IL-18 (1:500, Proteintech, China), rabbit anti-NLRP3(1:500; Proteintech, China), rabbit anti-ASC (Proteintech, China), rabbit anti-GAPDH (Proteintech, China) antibodies overnight at 4 °C. The blots were washed three times and incubated for 1 h with horseradish peroxidase HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000; Proteintech) in TBST. The blots were then washed in TBST, and bands were visualized using an enhanced chemiluminescence system (Advansta, USA) and the ChemiDoc Touch Imaging System (Bio-Rad). Relative protein levels were determined by normalization to the GAPDH signal using Image lab software (Bio-Rad, CA, USA).

4-Hydroxytamoxifen was purchased from Aladdin (Shanghai, China). Jasplakinolide, taxol and caffeine were obtained from Abcam (Cambridge, UK). 2-Aminoethyl diphenylborinate, dantrolene, Z-VAD-FMK, SP600125, lipopolysaccharide (LPS) and nigericin were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MCC950 were obtained from CSNpharm (Chicago, IL, USA). PEG8000 and PF431396 were obtained from Beyotime Biotechnology (Shanghai, China). Cantharidin was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Rabbit anti-caspase-1, rabbit anti-ASC and rabbit anti-NLRP3 antibodies were obtained from Proteintech (Chicago, IL, USA). Mouse anti-α-tubulin antibody was from Boster (BM1452, Wuhan, China). Mouse anti-β-actin was purchased from Cell Signaling Technology (Danvers, MA, USA). The siRNA targeting ASC and CASPASE-1 was constructed by GenePhrama (Shanghai, China). The Flag-Gsdmd-NT, Flag-Gsdmd-CT plasmid were purchased from Addgene (Watertown, MA, USA).