Figure 5.
SCFCdc4 inhibits PGC-1α activity. COS7 cells were transiently transfected with the indicated contructs and luciferase activity was measured and normalized to cotransfected βgal activity. Relative activity represents the average normalized luciferase activity of at least three independent experiments. Error bars represent the standard error of the mean. (A) ERRα coactivation assay: PGC1α (or vector control pcDNA3) and GST-Cdc4 (or vector control) were transiently transfected into COS7 cells with an ERRα-responsive luciferase reporter construct. (B) COS7 cells were transfected with plasmids expressing Gal4-DBD, Gal4DBD-VP16, or Gal4DBD-PGC1α, and the indicated isoforms of GST-Cdc4 (or vector control) with a Gal4-responsive luciferase reporter. (C) The indicated CPD mutants of Gal4-PGC1α were transiently cotransfected into COS7 cells together with a Gal4-responsive luciferase reporter construct and GST or GST-Cdc4α. 3A and 4A correspond to mutations in which alanines are substituted for the three p38 phosphorylation sites (T263, S266, T299) or all four phosphorylation sites (T263, S266, T295, and T299), respectively. (D) COS7 cells were cotransfected with Gal4 fusion constructs (Gal4-DBD, Gal4DBD-PGC-1α wild type, or the indicated mutants) and βgal. Cytoplasmic and nuclear extracts were harvested 48 h later. The amount of nuclear extract loaded on the gel was normalized for transfection efficiency based on βgal activity in the cytoplasmic extracts. Expression levels of Gal4 fusion proteins were determined by anti-PGC-1α Western blot.