Figure 5.

One of the two putative STAT5 binding sites in the HNF-3γ promoter could bind to bovine liver STAT5 in vitro. A, EMSA of the two putative STAT5 binding sites. In this assay, a ³²P-labeled double-stranded oligonucleotide corresponding to the putative STAT5 binding site 1 or site 2 (see Fig. 4A and Table 1) was incubated with liver nuclear protein extracts from +GH and −GH cattle followed by gel electrophoresis. B1 and B2 indicate two DNA-protein complexes. B, Competitive gel-shift assay of the putative STAT5 binding site 2. In this assay, the ³²P-labeled oligonucleotide corresponding to this STAT5 binding site was incubated with liver nuclear protein extracts from +GH cattle in the presence of 1×, 10×, and 100× molar excess of the same unlabeled oligonucleotide or an unrelated oligonucleotide. C, Supershift assay of the putative STAT5 binding site 2. In this assay, the ³²P-labeled oligonucleotide corresponding to this STAT5 binding site was incubated with liver nuclear protein extracts from +GH cattle in the presence of an anti-STAT5 antibody or an equal amount of rabbit preimmune serum. S1 and S2 indicate probable supershifts of the DNA-protein complexes B1 and B2, respectively. These assays were repeated at least two times; shown are representative results.