FIG. 3.

Supramolecular organization of respiratory Complex I and Complex III in R4B 1:1 and R4B 1:30 proteoliposomes. (A) R4B 1:1 and (B) R4B 1:30 samples were separated by 2D BN/SDS-PAGE after solubilization with digitonin at a detergent to protein ratio of 8 (w:w) and resolved by western blotting followed by immunodetection using monoclonal antibodies specific for single respiratory subunits. The images shown in the picture were obtained in camera by double overlaying exposures (not postproduction computer-graphic overlay) to the antibody against the NDUFA9 (39 kDa) subunit of Complex I and, in sequence, to the antibody against the Rieske protein (22 kDa) of Complex III. Arrows point to monomeric Complex I. The upper panel of the figures schematically shows the position of supercomplex I₁III₂ (SC), Complex I (CI) and dimeric Complex III (CIII) in the 1D BN-gel prior to second dimension electrophoresis (2D SDS-PAGE). NADH-cytochrome c and NADH-ubiquinone oxidoreductase activities are expressed in nmoles of NADH·min⁻¹·mg protein⁻¹. n indicates the number of independent samples. Data are given as mean±S.D. Numbers followed by asterisks are significantly different according to the Student's t-test (*p<0.05). R4B, mitochondrial fraction enriched in Complex I and Complex III.