Anti cyt c

Total protein from AGS and HGC27 cells was extracted using commercial kit, and the protein concentration was determined with BCA method. The samples were subjected to SDS–PAGE, and were transferred to the PDVF membrane that were placed in 5% BSA solution (ThermoFisher Scientific, MA, USA). The primary antibodies were incubated at four degrees overnight. The primary antibodies utilized in the experiments included anti-BAX (Proteintech, 50599-2-Ig, 1:1000), anti-β-action (Proteintech, 66009-1-Ig, 1:5000), anti-Bcl-2 (Proteintech, 68103-1-Ig, 1:1000), anti-PARP (Cell Signaling Technology, #9532, 1:1000), anti-Cleaved PARP (Cell Signaling Technology, #5625, 1:1000), anti-Caspase-3 (Cell Signaling Technology, #14220, 1:1000), anti-Cleaved Caspase-3 (Cell Signaling Technology, #9664, 1:1000), anti-PI3K (Affinity, AF6241, 1:1000), anti-p-PI3K (Affinity, AF3242, 1:1000), anti-AKT (Cell Signaling Technology, #4691, 1:1000), anti-p-AKT (Cell Signaling Technology, #4060, 1:1000), anti-EGFR (Proteintech, 66455-1-Ig, 1:1000), Anti-Cyt C (Proteintech, 10993-1-AP, 1:1000). The secondary antibodies were added and incubated at room temperature for 1 h. Finally, the bands were visualized using the IMAGE J software, with β-action serving as the internal standard.

Crude proteins were extracted from HK-2 cells treated with AAⅠ. Western blot assay was carried out as previously reported 20 (link). The primary antibodies were used anti-Caspase 3 (Proteintech, USA), anti-Bax (Proteintech, USA), anti-Bcl-2 (Proteintech, USA), anti-Cyt C (Proteintech, USA) and anti-β-actin (Affinity biosciences, China). The protein bands were quantified by ImageJ software, and normalized to the corresponding to the β-actin level.