Western blot analysis of mitochondrial proteins.
A, 20 μg of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with eight respiratory complex subunits in six cell lines with TOM20 as a loading control. ND1, ND4, ND5, and ND6, subunits 1, 4, 5, and 6 of NADH dehydrogenase (complex I); CYTB, cytochrome b of ubiquinone cytochrome c oxidoreductase (complex III); CO2, subunit 2 of cytochrome c oxidase (complex IV); ATP6 and ATP8, subunit 6 and 8 of the H+-ATPase (complex V). B, quantification of eight respiratory complex subunits. Average relative values of ND1, ND4, ND5, ND6, CYTB, CO2, ATP6, and ATP8 were normalized to the average values of TOM20 in various cell lines. The values for the latter are expressed as percentages of the average values for the control cell line C9. The calculations were based on three independent determinations. C, Western blot analysis of Afg3l2 and Clpp proteins in six cell lines with β-actin as a loading control. Graph details and symbols are explained in the legend to Fig. 4. Error bars, S.D.