*3.6. Western Blot Analysis*

HCT116 cells (2 × 105 cells/mL) were placed in a 36-mm culture dish and incubated for 24 h. A variety of concentrations of SA were then added, with or without CoCl2, and the cells were cultured for the indicated time points before being digested. The protein was extracted with lysis buffer, and the protein concentrations were determined using the bicinchoninic acid (BCA) method. A 40 μg protein sample was collected from each group, boiled for 10 min, loaded onto 10% SDS-PAGE gels, and then transferred to PVDF membranes with electroblotting. Membranes were blocked for 1 h with 5% fat-free milk at room temperature, rinsed with PBS, and incubated with diluted primary antibodies 1:1000 or 1:500 overnight at 4 ◦C. Then, the membranes were incubated with specific secondary antibodies (1:1000) for 2 h and rinsed with PBS. The expression of β-actin was used as an internal standard. Proteins were detected with an enhanced chemiluminescence detection kit from GE Healthcare (Little Chalfont, UK) and an LAS-4000 Imager (Fuji Film Corp., Tokyo, Japan).
