The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPARα and HNF4α in the Liver

Figure 6

Binding of PPARα/RXRα to TB PPRE3 is not enhanced by HNF4α. (a) Schematic outline of the DAPA procedure. (b) DAPA was performed with nuclear extracts of transfected COS-7 cells (with either HNF4α, PPARα, or RXRα) using the biotinylated TB PPRE3 oligonucleotides. Complexed proteins were resolved by SDS-PAGE and revealed by Western blot using an anti-PPARα (Figure 6(b)) or anti-HNF4α antibody (Figure 6(c)). (d) Coimmunoprecipitation of RXRα and HNF4α. Nuclear extracts from transfected COS-7 cells were immunoprecipitated (IP) with PPARα or RXRα antibodies, as indicated. The total plasmid amount was adjusted with pCDNA3.1 parent vector to 8 μg for each 100-mm transfection with Exgen 500. Cells were incubated in DMEM 10% FCS with 10 μM Wy for 48 h. The presence of HNF4α protein in the immunopurified material (100 μg of nuclear protein) was detected by Western blot assay using anti-HNF4α antibody. NE: Nuclear Extracts. Lane 1: Only 4 μg of nuclear protein were loaded.