Fig. 2.

CR promotes the assembly of mitochondrial supercomplexes. a Blue native polyacrylamide gel electrophoresis (BN-PAGE) of liver tissue samples from 12-month-old mice fed AL or 30 % CR. Coomassie blue staining of 1D BN-PAGE gel (left panel) and subsequent immunoblotting with anti-Cox6b1 antibody (right panel). SDS-PAGE and immunoblotting with anti-VDAC/Porin antibody was also performed to confirm equivalent protein loading of mitochondria lysates (lower panel). b Densitometric analysis of immunoblots. The results are presented as mean ± SD (n = 6, **p < 0.01, ***p < 0.001, vs. AL group). c 2D BN/SDS-PAGE of AL and CR liver samples. Immunoblotting was performed using the anti-ATP5A antibody for Complex V, the anti-Core2 antibody for Complex III, the anti-SDHB antibody for Complex II, and the anti-Cox1 and the anti-Cox6b1 antibodies for Complex IV. The position SC-a corresponds to the mitochondrial supercomplexes containing I/III₂/IV_n (n = 1, 2, 3, or 4); the position SC-b corresponds to I/III₂; The position SC-c corresponds to Complex V dimers. The positions of Complex V monomer (CV), Complex III monomer (CIII), Complex III dimers + Complex IV monomers or dimers (III₂IV_n, n = 1 or 2), Complex IV monomer or dimers (CIV or IV₂), and Complex II monomers (CII) are also indicated. CV and III₂IV_n spots are overlapping. The values represent the proportion of signal intensity in each spot in a row detected by a specific antibody