Nbt bcip reagent kit

HCT116/ATB and LOVO/ATB cells were washed with PBS buffer. Then, the cells were resuspended and lysed using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) to collect total protein. The concentration of proteins was measured by the bicinchoninic acid method. In total, 50 μg of protein was separated on a 12.5% sodium dodecyl sulfate-polyacrylamide-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Sigma-Aldrich). After blocking by 1% bovine serum albumin (BSA) for 1 hour, the PVDF membranes were incubated with the primary specific antibody of Bcl-2 (1 : 1,000; CST), caspase-3 (1 : 1,000; CST), GATA6 (1 : 1,000; CST), and GAPDH (1 : 1,000; CST) at 4°C for 24 hours. Then, the membranes were incubated with corresponding HRP-labeled Goat Anti-Rabbit IgG (H+L) (Abcam, Cambridge, UK). Finally, the PVDF membrane was stained using NBT/BCIP Reagent Kit (Thermo Fisher Scientific) and quantified using ImageJ software. GAPDH was used as an internal control. The experiment was done with three biological triplicates.