Figure 2.—

The amc mutants are deficient in complex I assembly. (A and B) Immunoblot analyses of membrane fractions, separated by BN-PAGE, using anti-49 kDa (A) and anti-51 kDa (B). A total of 150 μg of proteins was loaded per lane. The figures show the most representative blots of three independent experiments. The solid and shaded arrowheads indicate the position of mature complex I and the 700-kDa subcomplex, respectively. Equal loading was tested by Coomassie blue staining (not shown) and the presence of a nonspecific band revealed by the anti–49-kDa antiserum (Figure S2B for an example). (C) Membrane fractions were separated by SDS–PAGE and immunoblot analyses were performed using polyclonal antibodies against subunit-specific peptides. The subunits are 51 kDa, TYKY, and 49 kDa. A total of 10 μg of total protein per lane was loaded. Antibody specificity was evaluated by peptide competition assays (not shown). Only the top band is specific for the 49-kDa subunit. Plastid cytochrome f was used as a loading control. The results are representative from at least three independent membrane extractions.