Figure 3. AMPK activation increases GATA/EBox DNA binding.

A. Representative EMSAs of nuclear extracts from Vehicle− (−AIC) and AICAR− (+AIC) treated cells that were incubated with radiolabeled oligonucleotides corresponding to the EBox, SRE and IRS sequences found within the AICAR-responsive region of the PGC-1α promoter (as shown in Fig. 1). B. A representative EMSA (Left panel) and a summary of multiple experiments (Right panel) showing the effect of AICAR (A) on GATA/EBox-DNA binding. Values are represented as means±S.E.M (n = 8) relative to vehicle-treated (V) cells. C. Representative EMSAs of nuclear extracts that were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt. Vehicle-treated cells were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt as well as antibodies against MyoD, USF-1 and c-Myc which are known to bind to the EBox sequence. FP: free probe, 25×, 50×, 100× CO: 25-fold, 50-fold, 100-fold molar excess of cold oligo, No Ab: No Antibody, SRF: SRF antibody, FKHR: Forkhead antibody, GATA-4: GATA-4 antibody, c-Myc: c-Myc Antibody, USF-1: USF-1 antibody. **The representative blot of IRS/DNA binding was made from parts of the same gel. D. Representative chromatin immunoprecipitation from cells treated with or without 1 mM AICAR for 24 hours. Protein/DNA complexes were immunoprecipitated with USF-1 antibody, or with non-specific IgG. Primers encompassing the region between −473 and −823 were used to analyze USF-1 binding to the PGC-1α promoter. The representative blot on the left was made from parts of the same gel. At right is a graphical summary of repeated experiments. Values are representative of means±S.E.M (n = 6).