Cleaved gsdmd

The total protein of ileum tissues for western blotting was extracted, and the protein concentration was determined by a BCA protein assay kit following the manufacturer’s instructions. Then the protein was separated by SDS-PAGE. After being separated, the protein was transferred onto PVDF membrane. Subsequently, the membrane was blocked with 5% BSA, and incubated with primary antibodies. Here, Cleaved caspase-1 (Affinity, AF4005, United States), NLRP3 (Servicebio, GB114320, China), Cleaved GSDMD (Abcam, ab255603, United States) and GAPDH (Servicebio, GB11002, China) antibodies were used and blots were quantified using ImageJ Software (NIH, Bethesda, MD, United States). Each group was tested using three samples. The results were normalized to the GAPDH band. The standard procedure was the same as that used in our previous study (Yang X. et al., 2020 (link); Yang H. H. et al., 2020 (link)).

Cells or homogenized kidney tissues were harvested in radioimmunoprecipitation assay buffer (Beyotime) and fully lysed on ice. After centrifugation (12 000 rpm, 10 minutes), the supernatants were collected, and the protein concentration was determined. Equal amounts of proteins were separated by SDS‐PAGE and then transferred onto poly(vinylidene fluoride) (PVDF) membranes. The membranes were blocked with 5% nonfat milk/Tris buffered saline with Tween (TBST) and successively incubated with the corresponding primary and secondary antibodies. The protein signals were then assessed using enhanced chemiluminescence (Thermo Fisher Scientific). The following primary antibodies were used: ASC (sc‐514414), PKA (sc‐390548), P2X₇R (sc‐514962) and RXFP‐1 (sc‐293228) from Santa Cruz Biotech; cleaved caspase‐1 (4199/89332) and pro‐caspase‐1 (3866) from Cell Signaling Technology; IL‐1β (WL00891) and NLRP3 (WL02635) from Wanleibio (Wanleibio); cleaved GSDMD ([ab215203; Abcam], [sc‐393656; Santa Cruz Biotech]); IL‐18 (10663‐1‐AP; ProteinTech); and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (AC002; ABclonal). Peroxidase‐conjugated secondary antibodies (ZSGB‐BIO) were used.