Defining a hierarchy of binding affinities of Stat5b for individual DNA sites within the rat <i>Igf1</i> locus. A

<p>. Gel-mobility shift experiments were performed with the Cy5.5-labeled double-stranded probe R34, 2 µg of nuclear protein from Cos-7 cells transfected with expression plasmids for the mouse GH receptor and rat Stat5b, and incubated with rat GH [40 nM] for 1 h, and various concentrations of competitor DNAs as indicated. Two representative individual competition experiments are shown. The arrow indicates the location of protein-DNA complexes (NS, no Stat5b in nuclear protein extract, FP = unbound probe). <b>B</b>. The graph illustrates results of competition experiments for 4 different unlabeled double-stranded competitor DNAs (mean ± S.E., n = 3 independent experiments, with 4 data points/experiment). <b>C</b>. Results for all probes have been tabulated (n = 3 independent experiments, with 4 data points/experiment) and are presented as IC50 values (DNA concentration at which binding of labeled probe is reduced to 50% of starting value). The 95% confidence interval (CI) also is indicated and each Stat5b core DNA binding sequence is listed.