Figure 2.

Two putative HNF-3 binding sites in the bovine IGF-I promoter could bind to bovine liver HNF-3γ protein in vitro. Panel A, EMSA of the putative HNF-3 binding sites. In this assay, a ³²P-labeled double-stranded oligonucleotide corresponding to each of the three putative HNF-3 binding sites (see Fig. 1A and Table 1) was incubated with liver nuclear protein extracts from cattle before (−GH) and after (+GH) GH injection, followed by gel electrophoresis. B indicates a DNA-protein complex. Panel B, Competitive gel-shift assay of the putative HNF-3 binding site 1. In this assay, the ³²P-labeled oligonucleotide corresponding to the putative HNF-3 binding site was incubated with GH-treated liver nuclear protein extracts in the presence of 1×, 10×, and 100× molar excess of unlabeled the same oligonucleotide or an unrelated oligonucleotide. Panel C, Supershift assay of the putative HNF-3 binding site 1. In this assay, the ³²P-labeled oligonucleotide was incubated with GH-treated liver nuclear protein extracts in the presence of an anti-HNF-3α, anti-HNF-3β, or anti-HNF-3γ antibody or an equal protein amount of goat preimmune serum. S indicates a supershift of the DNA-protein complex B. These assays were repeated at least two times; shown are representative results.