Horseradish peroxidase conjugated anti rabbit or anti mouse antibodies

Cells were washed with ice-cold PBS, lysed using a radioimmunoprecipitation assay (RIPA) buffer (Bio-solution, Suwon, Korea), and centrifuged at 12,000 × g for 15 min at 4°C. The protein concentration was then measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein extracts (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) and incubated overnight at 4°C with primary antibodies against phosphorylated (p)-AKT1/2/3, AKT1/2/3, cAMP response element–binding protein (CREB), p-CREB, BAX, HO-1, and β-ACTIN. Detailed information on these antibodies is listed in Supplemental Materials and Methods. The membrane was then washed three times with TBST for 15 min each and incubated for 1 h at room temperature with horseradish peroxidase–conjugated anti-rabbit or anti-mouse antibodies (1:10,000) (Abcam, Cambridge, UK). The immunoreactivity of the blot was examined using an enhanced chemiluminescence kit (Thermo Fisher Scientific). The electrochemical images were then obtained using a Davinci-K Gel Imaging System (Davinci-K, Seoul, Korea). The relative densities of bands were quantified using ImageJ software (Version 1.50; National Institutes of Health, Bethesda, MD, USA).