Fig. 3

Analysis of stability and activity of OXPHOS complexes. A, assembly and stability of OXPHOS complexes. Mitochondria extracted from various cell lines were solubilized with n-dodecyl-β-D-maltoside, electroblotted, and hybridized with antibody cocktail specific for subunits of five OXPHOS complexes and with TOM20 as a loading control. B, quantification of the levels of complexes I, II, III, IV, V and supercomplexes (SC) in mutant and wild-type cell lines. The calculations were based on three independent determinations. C, in-gel activity of respiratory chain complexes I, II, and IV. A total of 20 μg of mitochondrial proteins (8 g/g digitonin/protein ratio) from various cell lines was used for BN-PAGE, and the activities of complexes were measured in the presence of specific substrates (NADH and NTB for complex I, sodium succinate, phenazine methosulfate, and NTB for complex II, DAB and cytochrome c for complex IV). Coomassie staining was used as a loading control. D, enzymatic activities of respiratory chain complexes. The activities of respiratory complexes were investigated by enzymatic assay on complexes I, II, III, IV, and V in mitochondria isolated from various cell lines. The calculations were based on three independent experiments. Graph details and symbols are explained in the legend to Figure 2.