Assessing binding affinity of Stat 5b for individual DNA sites.

<p>Quantitative DNA-protein binding was assessed by gel-mobility shift experiments as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050278#s2" target="_blank">Materials and Methods</a>’ using varying concentrations of Cy5.5-labeled double-stranded oligonucleotides for R58, R34, or R35, and 1 µg of nuclear protein from Cos-7 cells transfected with expression plasmids for the mouse GH receptor and wild-type rat Stat5b, and incubated with rat GH [40 nM] for 1 h. DNA binding was quantified with a LiCoR Odyssey infrared scanner and v3.0 analysis software, and results were plotted as shown. <u>Left panels</u>: representative results from individual experiments using nuclear proteins from cells expressing the mouse GH receptor and wild-type Stat5b after GH treatment. FP = unbound probe. The arrow indicates protein-DNA complexes. <u>Right panels</u>: binding curves with Kds listed (mean ± S.E., n = 3 experiments).