Figure 2. Mammalian SDHAF4 is required for maximal SDH activity.
(A) C2C12 cells were transfected with either a control non-targeting siRNA or either of two siRNAs directed against SDHAF4 (si1 and si2), and SDHAF4 mRNA abundance was assayed by quantitative RT-PCR and normalized to tubulin mRNA (±SEM. N=3 biological replicates. ***p < 0.0005). (B) SDH enzyme activity was measured in mitochondria harvested from C2C12 cells treated as in (A) (±SEM. N=3 biological replicates. ***p < 0.0005; *p < 0.05). (C) Mitochondria extracted C2C12 cells treated as in (A) were solubilized in digitonin, fractionated by BN-PAGE, and analyzed by immunoblotting for ATP5A (complex V monomers (CVI) or dimers (CVII), QCR2 (complex III), VDAC, or SDHA (complex II).