Figure 6.

Adiponectin (APN)-mediated increase in the binding activity of PPARα to the PPRE of the heme oxygenase (HO)-1 promoter region by an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). A: Cells were cultured and pretreated with a PPARα or AMPK antagonist for 1 hour before the addition of 30 μg/ml APN for one hour, with Wy14643 used as a positive control. The putative PPRE-binding activity derived from the HO-1 promoter region of nuclear proteins was assayed by EMSA in cells with the indicated treatments. 100× cold denotes a 100-fold molar excess of unlabeled oligonucleotides relative to the biotin-labeled probe; this was added to the binding assay for competition with the unlabeled oligonucleotide. The mobility of specific PPARα complexes is indicated. B: Cell lysate was subjected to a ChIP assay. The DNA associated with the PPRE was immunoprecipitated with an anti-PPARα antibody, and PCR amplification was used to determine the extent of PPARα association with the functional PPRE in an HO-1 promoter fragment of 162 bp. Distilled water (ddH2O) and anti-GAPDH were used, respectively, as negative controls for the PCR and ChIP assays. Representative results of three separate experiments are shown, and data are presented as the mean ± SEM (*P < 0.01 vs. the control; **P < 0.01 vs. APN alone).