Fig. 6. Deacetylation of mitochondrial proteins and CypD by NAM treatment.

Acetylation levels of total mitochondrial proteins and CypD were determined. (A) Mitochondrial fractions were obtained from either fibroblasts or H460 cells, cultured in the presence of NAM for 1, 2, or 3 days, and equal amounts of protein were applied for Western blot analyses with antibodies against acetylated-lysine (Ace-K), CypD, or Tom20. Arrows in Ace-K blots point to the band that migrated to the same position as the CypD band in the CypD blots. The intensities of the bands pointed by the arrow, relative to the intensities of CypD bands of the same time points, were determined through densitometry and were noted under the panels of the Ace-K blot. (B) Total cell lysates were probed with anti-CypD antibody overnight at 4°C, and were further incubated with protein A/G plus-agarose beads. Precipitated proteins were analyzed by Western blotting with anti-acetylated-lysine antibody. The filter was then stripped and reprobed with anti-CypD to show levels of total CypD.