Figure 2.

Altered assembly and defective stability of complex I. (A) Respiratory complex I assembly. Whole cells from mutant and control cell lines were solubilized with digitonin and then subjected to BN-PAGE analysis. Blots were then hybridized with NDUFA9 (complex I, CI), SDHA (complex II, CII), UQCRC2 (complex III, CIII), and CO2 (complex IV, CIV) antibodies, with ATP5A (complex V, CV) as the internal control. (B) Quantification of complexes. The levels of four complexes in the mutant and control cell lines were determined based on three independent determinations in each cell line. (C) In-gel activity of complexes I, II, and V. Mitochondrial proteins (20 µg) from various mutant and control cell lines were used for BN-PAGE, and the activities of the complexes were measured in the presence of specific substrates (NADH and NBT for complex I; sodium succinate, phenazine methosulfate, and NBT for complex II; ATP and lead nitrate for complex V). (D) Quantification of in-gel activities of complexes I and II. The calculations were based on three independent determinations in each cell line. Graph details and symbols are explained in Figure 1.