Anti bak antibody

293T cells were washed with PBS and lysed with PBS containing 1% NP-40. Protein concentration was determined using Pierce BCA reagent (ThermoFisher; 23221, 23224). 35 μg protein was loaded in each lane of 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose membrane and immunoblotted with the following primary antibodies: anti-Bax antibody (Santa Cruz N20), anti-Bak antibody (Cell Signaling 3814), anti-Bid antibody (R&D Systems AF860), anti-Bim antibody (Sigma B7929), anti-Puma antibody (Cell Signaling 4976), anti-Bcl-2 antibody (Abcam 32124), anti-Bcl-xL antibody (Cell Signaling 2764), and anti-Mcl-1 (Santa Cruz S19) at 1:1,000 dilution. The secondary anti-rabbit and mouse antibodies, conjugated with HRP, were obtained from Santa Cruz and were used at 1:2,000 dilution. The luminescence signal was detected using ECL reagent (ThermoFisher 32106).

ARS and DHA were purchased from Holleypharm (Chongqing, China). FTS was purchased from Santa Cruz Biotechnology (California, USA). DHA, ARS and FTS solutions were prepared by dissolving them in dimethylsulphoxide (DMSO, Sigma, USA) before experiments. The final concentration of DMSO was less than 1% in all experiments. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). 2′, 7′-dichlorodihydrofluorescein diacetate (DCF-DA) and N-acetyl-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, USA). Mito-Tracker Deep Red 633 was purchased from Invitrogen (Massachusetts, USA). Ac-DEVD-CHO and Z-VAD-FMK were purchased from Beyotime (Jiangsu, China). Z-IETD-FMK was purchased from Selleckchem (USA). Mouse monoclonal anti-Bak antibody (Ab-2) and anti-Bax antibody (6A7) were purchased from Calbiochem (San Diego, CA, USA). Rabbit monoclonal anti-Bid and mouse monoclonal anti-Tubulin, anti-Bax and anti-Bak antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Proper goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Invitrogen (Massachusetts, USA). Turbofect^™ transfection regent was purchased from Thermo Fisher Scientific (Massachusetts, USA).