Western blot analysis

Antibodies against caspase-3 (cat. no. MAB4703), cleaved caspase-3 (cat. no. AB3623), caspase-8 (cat. no. MAB4708), poly ADP ribose polymerase (PARP; cat. no. AM30), cleaved PARP (AB3620) were purchased from Merck KGaA (Darmstadt, Germany); caspase-9 (cat. no. sc-8355) and apoptosis inducing factor (AIF; cat. no. sc-5586) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); Antibodies against pro-caspase-3 (cat. no. ab32499), pro-caspase-12 (cat. no. ab8117), caspase-12 and cleaved-caspase-12 (cat. no. ab18766), GAPDH (cat. no. ab9485) was obtained from Abcam (Cambridge, UK); cytochrome c antibody (cat. no. 556433) was purchased from BD Biosciences. Horseradish peroxidase (HRP)-labeled goat anti-mouse (cat. no. A0216) and goat anti-rabbit immunoglobulin G (cat. no. A0208) secondary antibodies were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).

Prior to protein extraction, cells were treated with CBDCA (0.04 or 0.08 mg/ml) with or without GSH (5 mM) for the indicated duration. Using RIPA lysis buffers (Sigma-Aldrich; Merck KGaA), total protein was extracted from HN-3 cells. Equivalent amounts of protein (100 µg/lane), whose concentration was determined by a BCA kit (Pierce; Thermo Fisher Scientific, Inc.), were loaded onto 10% polyacrylamide gels, subjected to electrophoresis, and transferred onto polyvinylidene fluoride membranes. Following blocking with 5% skim milk for 1 h at room temperature, primary antibodies were incubated overnight at 4°C [cytochrome c (1:200); AIF (1:200); GAPDH (1:2,000); PARP (1:200); cleaved-PARP (1:200); caspase-3 (1:1,000); cleaved caspase-3 (1:1,000); pro-caspase-3 (1:1,000); caspase-9 (1:500); caspase-8 (1:500); caspase-12 (1:500); pro-caspase-12 (1:500)]. Each membrane was probed with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG antibody (1:1,000) for 1 h at room temperature. Labeled protein bands were visualized by an ECL + plus western blotting system kit (cat. no. RPN3352; GE Healthcare Life Sciences, Little Chalfont, UK) and were detected by a Kodak in vivo image analyzer (Kodak, Rochester, NY, USA). The bands were analyzed by densitometry using ImageJ version k1.45 (NIH, Bethesda, MD, USA).