Figure 6

Trans -activation ability of PPARδ2 investigated by transient transfection of HeLa cells. A. Co-transfection of pFABPLuc reporter construct (200 ng) with expression vector for either PPARδ1 (100 ng), PPARδ2 (100 ng) or irrelevant plasmid DNA pUC19 (100 ng), untreated (control) or treated with 1 nM, 10 nM or 100 nM of the PPARδ specific ligand GW501516. B. Co-transfection of pFABLuc reporter construct (200 ng) with constant amount of expression vector for PPARδ1 (50 ng) and increasing concentrations of the expression vector for PPARδ2 (50 to 200 ng) and treatment with 10 nM or 100 nM GW501516. Transfection of the pFABLuc vector alone was used as a control. Plasmid DNA pUC19 was added to ensure equal amount of DNA in all transfections. Plasmid pSV-β-galactosidase control vector (Promega) (260 ng) was co-transfected in all experiments for normalization of the transfection efficiency. The data presented are the mean (± SD) luciferase/β-galactosidase ratios of three independent transfections determined in quadruplicates. The activities are expressed as relative values setting the value of untreated control to 1 (A) or the value obtained without PPARδ2 expression plasmid to 1 (B). C. Western blot analysis with nuclear extracts prepared from untransfected HeLa cells (Contr) and HeLa cells transfected with the expression vectors encoding PPARδ1 or PPARδ2, respectively, using a PPARδ antibody raised against the N-terminal region of the nuclear receptor (sc-7197, Santa Cruz Biotechnology). The sizes of standard molecule markers are given on the left side.