Ab321124

The protein sample was added to the corresponding proportion of SDS gel loading buffer and boiled for 5 min. After SDS-PAGE electrophoresis and transferred membrane, the membrane was blocked 5% skim milk in PBST buffer, at room temperature for 1 hour and washed with PBST for 3 times. Then ERK1/2, p-ERK, p38, p-p38, JNK, p-JNK, and Bcl-2 (ab321124, Abcam), Bax (ab53154, Abcam), and Caspase3 (ab44976, Abcam) (1:500) primary antibody were added, and the membranes were incubated overnight at 4°C, washed 3 times with PBST, and incubated in the corresponding secondary antibody labeled with horse. Then the membranes were added by radish peroxidase and incubated for 1 hour at room temperature. The membrane was washed 3 times and ECL developed. The gel imaging system was photographed, and the image J software was used to analyze the gray value.

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.