Western blot analysis and caspase-3/8 activity assay

The transfected cells were lysed with modified RIPA buffer at 4°C for 30 min. Protein concentration was determined using the BCA protein determination kit (Beyotime Institute of Biotechnology), and 30–50 µg of the protein samples were electrophoresed on 8–12% SDS-PAGE gels and transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). Subsequently, 5% milk was used to block the membranes in a shaker at 37°C for 1 h. Antibodies for Bax (cat. no. sc-6236, 1:1,000), phosphorylated Akt (cat. no. sc-7985-R, 1:1,000), PI3K (cat. no. sc-7174, 1:1,000), NF-κB (p65, cat. no. sc-109, 1:1,000) and GAPDH (cat. no. sc-25778, 1:5,000) antibodies, all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) were used for incubation at 4°C overnight. The membranes were then washed with TBST buffer and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (cat. no. sc-2004, 1:5,000, Santa Cruz Biotechnology) at 37°C for 1 h. The protein bands were visualized by ECL (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

For the analysis of caspase activity, total protein (3–5 µg) was incubated with caspase-3 and caspase-8 activity kits (Beyotime Institute of Biotechnology) at 37°C for 1 h. Caspase-3/8 activity was measured using a VERSAmax microplate reader (Molecular Devices, LLC) at 405 nm.