Gp91phox

Total protein was separated from hearts or cells, separated by SDS‐PAGE, and transferred to PVDF membranes. After being blocked with 5% non‐fat dry milk at room temperature (RT) for 2 hours, the protein was incubated with antibodies against Cleaved caspase‐3 (1:1000), caspase‐3 (1:1000), Bcl‐2 (1:1000), Bax (1:1000), gp91^phox (1:500), Cyt‐Cyto C (1:1000), NQO1 (1:1000), Nrf2 (1:1000), Sirt1 (1:1000), Ac‐Foxo1 (1:500), Foxo1 (1:1000), PI3K (1:1000), p‐Akt (1:1000), Akt (1:1000) and β‐actin (1:1000) at 4°C for 14 hours. After washed with TBST buffer (50 mmol\L Tris‐HCl, 150 mmol\L NaCl, 0.1% Tween 20, pH 7.6) for three times, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies (1:5000) at room temperature for 2 hours. Immunocomplexes were detected with an enhanced chemiluminescent protein detection kit, and bands were quantified by scanning densitometry with an image analyzer (BandScan, USA). Primary antibodies against Sirt1, PI3K, p‐Akt, Akt, Cleaved caspase‐3, caspase‐3, Bcl‐2 and Bax were from Cell Signaling Technology (Boston, MA, USA); antibodies against gp91^phox and β‐actin were from Santa Cruz Biotechnology; Ac‐Foxo1 and Foxo1 were from Medchem Express (NJ, USA). Secondary antibodies, including rabbit anti‐goat, goat anti‐mouse and goat anti‐rabbit antibodies, were from Zhongshan Co. (Beijing).

Whole-cell extracts were prepared using a low-salt lysis buffer extracts (150 mM NaCl and 0.1% NP-40) as previously described (Pardo et al., 2010). Samples were separated by PAGE (NuPAGE Bis-Tris gels; Life Technologies) and analyzed by Western blotting. The following antibodies were used: gp91phox (sc-130543; Santa Cruz Biotechnology, Inc.), p22phox (sc-20781; Santa Cruz Biotechnology, Inc.), β-actin (Abcam, ab75186), and Rac1/2/3 (PA5-17159; Thermo Fisher Scientific). Blots were developed using ECL Prime reagents (GE Healthcare) and Lumigen TMA-6. Lysis in RIPA buffer produced similar results.