Anti gsk 3b

Ovarian cancer cell lysates were prepared with RIPA lysis buffer (Beyotime, China) containing protease inhibitor cocktail (Roche). Protein samples were loaded for SDS/PAGE and transferred on to a nitrocellulose membrane. After a blockage with 5% fat-free milk, the membrane was probed with primary anti-β-catenin (dilution 1:3000, Santa Cruz Biotechnology), anti-cyclin D1 (dilution 1:1000, Santa Cruz Biotechnology), anti-cmyc (dilution 1:3000, Santa Cruz Biotechnology), anti-p-GSK-3b (dilution 1:1000, Santa Cruz Biotechnology), anti-GSK-3b (dilution 1:2000, Santa Cruz Biotechnology) and anti-GAPDH (dilution 1:5000, Santa Cruz Biotechnology) antibody. After washing, the membrane was incubated with horseradish peroxidase conjugated (HRP) secondary antibody (1:10000, Santa Cruz Biotechnology) for 1 h. The signal was visualized using the ECL detection system (Thermo Fisher, U.S.A.) and quantitated by densitometry using Quantity One software (Bio–Rad, Hercules, CA, U.S.A.).

Anti-Nrf2 (cell signaling, Lot: 12721), Anti-p-NFkB (Cell signaling, Lot: 3033), Anti-NF-κB (Cell signaling, Lot: 8242), Anti-p-GSK3b (Santa Cruz, Lot: sc-11757), Anti-GSK3B (Santa Cruz Lot: sc-9166), Anti-p-Akt (Cell signaling, Lot: 4060), Anti-Akt (Cell signaling, Lot: 4691), Anti-HO-1 (Cell signaling, Lot: 70081), Anti-Sirt1 (Cell signaling, Lot: 8469), Anti-GAPDH (Cell signaling, Lot:5174), and Anti-Tubulin (Santa Cruz, Lot: sc-8035).

Samples of the protein fractions containing equivalent amounts of proteins per lane (70 μg per lane) were separated by 10% or 12% SDS-PAGE gel electrophoresis (according to molecular weight of specific protein). For Western blot assays, proteins were transferred to a nitrocellulose membrane. The quality of the transfer was controlled by Ponceau S staining of nitrocellulose membranes after the transfer. Specific anti-Akt 1/2/3 (dilution 1:330), anti-p-Akt 1/2/3 (dilution 1:710), anti-eNOS (dilution 1:200), anti-PKCε (dilution 1:1000), anti-GSK3B (dilution 1:330), anti p-GSK3B (dilution 1:250), anti-BAX (dilution 1:200), anti-Bcl-2 (dilution 1:200) and anti-caspase 3 (dilution 1:100) (Santa Cruz Biotechnology) antibodies were used for the primary immunodetection. Peroxidase-labeled anti-rabbit immunoglobulin (Cell Signaling Technology) a peroxidase-labeled anti-mouse immunoglobulin were used as the secondary antibodies (dilution 1:2000). Bound antibodies were detected by the enhanced chemiluminescence (ECL) method. The optical density of individual bands was analyzed by PCBAS 2.08e software and normalized to GAPDH (anti-GAPDH antibody, dilution 1:750) as an internal control.