Protein Expression Analysis in H9c2 Cells

The protein expression of Akt, Bax, and Bad was evaluated as described previously with a slightly modification (Phosri et al., 2017 (link)). After treatment, H9c2 cells were solubilized in Triton X-100 lysis buffer (20 mM Tris pH 7.4, 0.8% Triton X-100, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 100 μM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail). After centrifugation, amount of protein in cell lysates was measured by a protein assay kit (Bio-Rad) and used bovine serum albumin as a standard. Samples were mixed with 4× SDS loading buffer and denatured by heating at 95°C for 5 min. After that, samples were subjected to SDS-PAGE gels and transferred to PVDF membrane (Bio-Rad), and separately immunoblotted with several antibodies such as Akt (Cell Signaling), phospho-Akt (Cell Signaling), Bax (Cell Signaling), Bad (Cell Signaling), and GAPDH (SantaCruz). Immunoblots were visualized with horseradish peroxidase-conjugated secondary antibodies and a SuperSignal chemiluminescent detection system (Thermo Scientific), using GAPDH as a loading control. The density of the band was calculated using ImageJ software.