P47phox

For western blot analysis, equal amounts of protein were separated by 4−12 % Bis-Tris Nu-PAGE gel and transferred to polyvinylidenedifluoride membranes. The membranes were blocked with 5 % nonfat milk and incubated with rabbit antibodies (1:1000) against Iba-1, p47^phox, gp91^phox (BD Transduction Laboratories), GAPDH, or rat CD11b antibody overnight at 4 °C. The next day, membranes were incubated with HRP-linked secondary anti-rabbit or rat IgG (1:3000) for 2 h at room temperature. ECL reagents (Amersham Biosciences) were used as a detection system.

Mouse left ventricle heart tissues were collected and homogenized in lysis buffer. The homogenates were centrifuged at 16 000 g at 4°C for 15 minutes. The BCA protein assay kit (Pierce Co) was used to analyse the protein concentrations. Equal amounts (20 µg) of the protein were separated by 10% SDS‐PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% non‐fat dry milk in Tris‐buffered saline and incubated with the following primary antibodies overnight: SIRT3 (1:1000; Cell Signaling), β‐MHC (1:1000; Abcam), TGF‐β1 (1:1000), gp91^phox and p47^phox (1:1000; BD transduction). After washing, the membranes were incubated for 2 hours with an anti‐rabbit or anti‐mouse secondary antibody coupled to horseradish peroxidase (1:5000; Santa Cruz). Densitometric analyses were carried out with image acquisition and analysis software (Bio‐Rad).