A22523

In kidney tissues and HK-2 cells, RIPA lysis buffer (20–188, Sigma-Aldrich) was used for extracting proteins. An equal quantity of protein was electrophoresed using SDS-PAGE and then transferred onto PVDF membranes. At 4 °C, primary antibodies against ELF4, Nrf2, NQO-1, GRP78, KIM-1, and IL-6 (1:1000, ab96075, ab137550, ab34173, ab21685, ab302932, and ab259341, Abcam, USA); IL-1β (1:1000, sc-12742, Santa Cruz Biotechnology); IL-18 and N-GSDMD (1:1000, A1115 and A22523, ABclonal, USA); Bax, Bcl-2, TNF-α, caspase-4, caspase-11, Caspase-12, CHOP, HO-1, and GSDMD (1:1000, 2772, 3498, 3707, 4450, 14340, 35965, 2895, 43966, and 39754, Cell signaling Technology, USA), and GAPDH (1:5000, 5174, Cell signaling Technology, USA) were deal with membranes overnight. After that, secondary antibodies (1:50000, 7074, Cell signaling Technology) were handled with membranes for 1 h. At last, protein bands were visualized by way of an enhanced chemiluminescence detection kit (Beyotime Biotechnology, China). Image J software (USA) was used to quantify protein expression.

GIST-T1 and GIST-T1 IR cell proteins were extracted using radioimmunoprecipitation assay buffer (AS1004; ASPEN) and evaluated using a bicinchoninic acid assay kit (AS1086; ASPEN). Average protein concentrations were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Then, the membrane was blocked in 5% skim milk for 2 h at room temperature and incubated in specific antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab181602, 1:10,000 dilution; Abcam), ANO6 (20784-1-AP, 1:1,000 dilution; Wuhan Sanying Biotechnology), Bax (50599-2-Ig, 1:2,000 dilution; Wuhan Sanying Biotechnology), Bcl-2 (ab321124, 1:1,000 dilution; Abcam), SLC7A11 (26864-4-AP, 1:1,000 dilution; Wuhan Sanying Biotechnology), SLC3A2 (15193-1-AP, 1:5,000 dilution; Wuhan Sanying Biotechnology), GSDMD-N (A22523, 1:1,000 dilution; abclonal), or cleaved-Caspase1 (AF4005, 1:500 dilution; affbiotech) overnight at 4°C. Membranes were then incubated with secondary antibodies (AS1107, 1:10,000 dilution; ASPEN) for 1 h. Finally, signals were developed using electrochemiluminescence detection system reagents (AS1059; ASPEN) according to the manufacturer’s instructions.