Figure 3.
GR–Stat5 interaction is dependent on the Stat5b N terminus. (A, top) Liver extracts were prepared from untreated or GH-injected mice, followed by quantitative α-Stat5b immunoprecipitation (I.P.) and subsequent immunoblotting with anti-P-Y-Stat5 antiserum. (Bottom) Blots were stripped and reprobed with anti-Stat5b-specific antiserum. Stat5b and Stat5bΔN proteins are fully tyrosine-phosphorylated upon GH stimulation. The amounts of total Stat5b were reduced to ∼40% in Stat5ΔN mice. (B) In vivo binding of Stat5 in proximal enhancers of ALS, Socs2, and IGF-1. The fold enrichment for each DNA fragment upon immunoprecipitation by anti-Stat5 followed by quantitative PCR is illustrated as histograms (mean ± SEM). Upon GH stimulation, wild-type Stat5b is highly bound to chromatin. Stat5bΔN displays ∼50%–70% of the DNA-binding capacity of full-length Stat5b. (C) The N terminus of Stat5b is essential for the GR–Stat5b interplay. Reciprocal immunoprecipitation of liver extracts using anti-Stat5b (left) or anti-GR (right) antisera. (Left) Stat5bΔN as well as full-length Stat5b proteins are efficiently immunoprecipitated with anti-Stat5b antisera (bottom), but only wild-type Stat5b interacts with the GR as shown via co-IP experiments (top). (Right) GR proteins are comparably pulled down (bottom), but only full-length Stat5b is coimmunoprecipitated (top). Four similar experiments were performed and one representative analysis is shown.