FIGURE 3.

GCN5 is required for the acetylation of p65K310 in skeletal muscle. A, bicistronic DNA constructs encoding shRNA against GCN5 and GFP were electroporated into the TA muscles, and mice were deprived of food for 2 days and fiber cross-sectional areas were measured. Representative images showing the electroporated (GFP-labeled) and non-electroporated fibers (marked by asterisks) (right image). Scale bar, 15 μm. B, GCN5 knockdown reduced p65K310 acetylation without a change in p300 content. Muscle lysates were used to determine the levels of the indicated proteins by Western blot. C, after immunoprecipitation of PGC-1α using the same muscle lysates of Fig. 3B, levels of acetylation were examined by Western blots. IP, immunoprecipitation. D, GCN5 overexpression in skeletal muscles stimulated by p65K310 acetylation. Wild type GCN5 was overexpressed in TA muscles, and mice were fed or fasted for 2 days. The same amounts (20 μg) of muscle extracts from each animal were analyzed for p65K310 acetylation and total p65 by Western blot (WB). Intensity of dots was quantified using ImageJ (National Institutes of Health). n = 4; *, p < 0.05. E, HEK293 cells were transiently transfected with the constructs in combinations as indicated above the each panel. Acetylation of p65 was determined by antibody to acetylated p65K310.