Anti procaspase 8

Total protein was extracted and protein concentrations established via bicinchoninic acid (BCA) assay. Protein (25 μg) was denatured, separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking overnight at 4°C using 5% BSA, the membranes were incubated with primary antibodies (anti-procaspase-8 1:2,500 and procaspase-9 1:2,000; both from Abcam, Cambridge, MA, USA), p-Akt 1:800 (Bioworld, St. Louis Park, MN, USA) for 2 h at room temperature, washed by TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at 1:2,000 dilution for 2 h. Bands were visualized using enhanced chemiluminescence (ECL; Applygen, Beijing, China) detection reagents and scanned images were quantified using ImageJ software. Experiments were performed in triplicate with β-actin used as a housekeeping control for normalization. The ratio of target gene to β-actin was used for semi-quantification and comparison between different groups.

Protein was extracted via RIPA lysis buffers (Beyotime, China), which was assessed with a bicinchoninic acid protein assay kit (Beyotime). The sample was separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. Then, the membrane was blocked using 5% non-fat milk for 1 h, which was incubated overnight with primary antibodies at 4°C and secondary antibody (1:2,000; Abcam, USA) lasting 1 h at room temperature. The primary antibodies included anti-Bax (1:1,000; Abcam), anti-Bcl2 (1:1,000; Abcam), anti-cleaved-caspase-3 (1:1,000; Abcam), anti-pro-caspase-3 (1:1,000; Abcam), anti-cleaved-caspase-8 (1:1,000; Abcam), anti-pro-caspase-8 (1:1,000; Abcam), anti-cleaved-caspase-9 (1:1,000; Abcam), anti-pro-caspase-9 (1:1,000; Abcam), and anti-GAPDH (1:1,000; Abcam). GAPDH served as a control. Image Lab™ Software (Bio-Rad, China) was used to quantify the intensity of blots.