P nrf2

Protein was extracted from frozen murine heart tissue or isolated cardiac myocytes using a Column Tissue and Cell Protein Extraction Kit (EpiZyme, China). BCA assay kits were used to measure the protein concentration. After boiling 10 min at 100 °C with loading buffer (EpiZyme, China), the protein samples were separated using 4–12% FuturePAGE™ gradient gels and MOPS-SDS running buffer (Nanjing ACE Biotechnology, China). The PVDF membrane (for iNOS detection, nitrocellulose membranes were used) was blocked with 5 % skimmed milk for 60–90 min, then incubated with primary antibody overnight at 4 °C. The secondary antibody was then added for 60 min at room temperature. An ECL regent (BioSharp, China) was used to visualize the protein bands using a gel imaging system (Bio-Rad Laboratories, Inc., USA). The following antibodies were used in the present study: iNOS (BD Transduction Laboratories™), Akt, nuclear factor erythroid 2–related factor 2 (Nrf2; Affinity Biosciences, China), phosphorylated (p)-Akt (Ser 473) (Wanleibio, China), NADPH oxidase 4 (NOX4), superoxide dismutase 2 (SOD2), p-Nrf2, translocase of outer mitochondrial membrane 20, heme oxygenase 1 (HO-1) (ProteinTech Group, Inc., China) and OXPHOS antibody cocktail (Abcam, UK).