Apaf-1 levels are decreased in myotubes.

Restoring Apaf-1 is sufficient to allow cytochrome c-mediated death. (A) Levels of the indicated apoptotic proteins were examined by Western blot of whole cell lysate from mC2C12 and dC2C12 cells. Tubulin serves as a loading control. Densitometry of protein levels are normalized to loading control protein levels of the representative Western blot. (B) Levels of the indicated apoptotic proteins were examined by Western blot of whole cell lysate from primary myoblasts and primary myotubes. Densitometry of protein levels are normalized to loading control protein levels of the representative Western blot. (C) RT-PCR was carried out with primers for the indicated mRNA using RNA from primary myoblasts and myotubes. GAPDH serves as a control. Densitometry of Apaf-1 mRNA levels are normalized to GAPDH levels of the representative gel. (D) dC2C12 cells were injected with plasmids for either Apaf-1 (Apaf) or empty vector as well as GFP. 24 h following injection, GPF positive cells were injected with rhodamine dextran and either yeast or bovine cytochrome c. Cell survival was assessed by morphology at the indicated times following cytochrome c injection. Data are the mean±SEM of n≥3 separate experiments per time point.