Anti bax

Diquat (Cat#: 6385-62-2) and melatonin (Cat#: 73-31-4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies anti-Sod1 (Cat#: WL01846), anti-Gpx1 (Cat#: WL02497a), anti-Bax (Cat#: WL01637), anti-Bcl2 (Cat#: WL01556), anti-Cleaved Caspase3 (Cat#: WL01992), anti-P53 (Cat#: WL01919), anti-ZO-1 (Cat#: WL03419), anti-Occludin (Cat#: WL01996), anti-β-catenin (Cat#: WL0962a), and anti-Connexin43 (Cat#: WL02837) were purchased from Wanleibio (Shenyang, China), while anti-Tubulin (Cat#: AF1216) was obtained from Beyotime (Shanghai, China). Unless otherwise indicated, the remaining reagents and chemicals used in this study were purchased from Sigma-Aldrich.

Cultured cells were harvested and lysed with NP-40 lysis buffer (Beyotime, Haimen, China) for total protein extraction, whereas xenograft tumor tissues were physically homogenized and lysed with RIPA lysis buffer containing 1% v/v PMSF (Beyotime). Proteins were separated by SDS-PAGE, and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% milk in TBST buffer (TBS + 0.05% v/v Tween-20), and probed for protein of interest with a specific primary antibody overnight at 4°C, followed by incubation with a secondary antibody for 1 h at room temperature (RT). Anti-YB-1 antibody was purchased from Cell Signaling (Boston, MA, USA), anti-cyclin D, anti-cyclin A, anti-cleaved caspase-3, anti-cleaved PARP, anti-Bcl-2 and anti-Bax antibodies were from Wanleibio (Shenyang, China); HRP-conjugated goat anti-rabbit IgG secondary antibody was purchased from Beyotime. Immune complexes were visualized using the ECL system (Qihai Biotech, Shanghai, China). To verify equal protein loading and transfer, membranes were stripped with stripping buffer (Beyotime) and re-probed with anti-β-actin antibody (Wanleibio).

H9C2 cells with different treatment were lysed, quantified and subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes (Cat # 1,620,177, Bio-Rad, Hercules, CA, USA) which were incubated with 5% nonfat milk in TBST and followed by incubation of primary antibodies at 4°C overnight. Then the PVDF membranes were washed with TBST and incubated with secondary antibodies. Protein expression was detected using BeyoECL Moon (Cat # P0018FS, Beyotime) under a Tanon 5200 machine (Shanghai, China). The detailed primary and secondary antibodies were listed as below: anti-cleaved caspase 3 (Cat # E83-77, Abcam); anti-caspase 3 (Cat # ab32351, Abcam); anti-Bax (Cat # WL01637, Wanleibio, Shenyang, China); anti-Bcl-2 (Cat # WL01556, Wanleibio); anti-β-actin (Cat # WL01372, Wanleibio); anti-Nrf2 (Cat # WL02135, Wanleibio); anti-Sirt2 (Cat # ab211033, Abcam); anti-HO-1 (Cat # EP1391Y, Abcam); anti-GST (Cat # ab138491, Abcam); anti-NQO1 (Cat # 11,451-1-AP, Proteintech, Wuhan, China); anti-FOXO3a (Cat # 66,428-1-Ig, Proteintech); anti-GCLM (Cat # 14,241-1-AP, Proteintech); anti-Keap1 (Cat # 10,503-2-AP, Proteintech). IgG(H + L)(HRP-labeled Goat Anti-Rabbit IgG(H + L)) and IgG(H + L)(HRP-labeled Goat Anti-Mouse IgG(H + L)) were purchased from Beyotime (Beijing, China). All protein expressions were normalized by β-actin expression.