Enhanced bicinchoninic acid assay kit

C₃H₁₀T_1/2 cells provided by the National Collection of Authenticated Cultures, Chinese Academy of Sciences were plated in a 6-well plate at a density of 8 × 10⁴ cells/well for 24 h and were pretreated with 0, 10^− 8, 10^− 7, or 10^− 6M Tanshinone IIA for 24 h. After incubation in 500µM H₂O₂ for another 24 h, the cells were collected, and total proteins were extracted on ice using radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China). The protein concentration was detected using an enhanced bicinchoninic acid assay kit (P0010, Beyotime Biotechnology, Shanghai, China). Western blot analysis was conducted as previously described [18 ]. The following antibodies were used: anti-cleaved caspase 3 (ab214430), anti-pro caspase 3 (ab32499), anti-superoxide dismutase 1 (SOD 1, ab13498), anti-heme oxygenase-1 (HO-1, ab68477), anti-catalase (CAT, ab16731) from Abcam (Cambridge, UK) and anti-Nrf2 (12721), anti-β-actin (8457), anti-Keap1 (8047), anti-Bcl2 (3498) and anti-Bax (5023) from Cell Signaling Technology (Danvers, Massachusetts, USA). A chemiluminescent horseradish peroxidase substrate kit (WBKLS0500, Millipore, Billerica, Massachusetts, USA) was used for the electrochemiluminescence detection assay.

The rabbit polyclonal immunoglobulin G (IgG) anti-CD95 antibody (N-18; cat. no. sc-714), mouse monoclonal IgG₁ anti-caspase-8 antibody (1.1.40; cat. no. sc-81656), rabbit polyclonal IgG anti-caspase-3 antibody (H-277; cat. no. sc-7148), goat polyclonal IgG anti-poly(ADP-ribose) polymerase 1 (PARP1) antibody (A-20; cat. no. sc-1562) and mouse monoclonal IgG₁ anti-β-actin antibody (C4; cat. no. sc-47778) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The radioimmunoprecipitation buffer and enhanced bicinchoninic acid assay kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Polyvinylidene difluoride (PVDF) membranes were purchased from EMD Millipore (Billerica, MA, USA). The LipoFiter™ Liposomal Transfection reagent was purchased from HanBio (Shanghai, China). The SP test kit and diaminobenzidine (DAB) colorization test kit were purchased from Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd. (Beijing, China). The AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit was purchased from KeyGen Biotech, Co., Ltd. (Nanjing, China).