Effect of STAT5b-RARα on STAT5b transcriptional and DNA binding activity.

(A) COS-7 cells were transiently transfected with the human prolactin receptor, a β-casein–luciferase reporter gene construct, and STAT5b with or without STAT5b-RARα. Transfected cells were stimulated without or with ovine prolactin (500 ng/mL) for 24 hours. Luciferase activity was measured and normalized for transfection efficiency using a β-galactosidase reporter construct. Data presented represent the mean ± SD of 3 separate experiments. (B) Gel-shift assays were performed using the PRE and WCEs of transiently transfected COS-7 cells that were incubated without or with prolactin (500 ng/mL) for 30 minutes. The location of the specific STAT5b/DNA complex is indicated with the solid triangle; a nonspecific band is indicated with the empty triangle. The level of protein expression, determined by immunoblotting with antibody against STAT5b, is shown in the bottom panel. (C) 293T cells were transiently transfected with the human β-casein–luciferase reporter gene construct and expression constructs for IL-2Rβ, γ_C, Jak3, and STAT5b with or without STAT5b-RARα. Transfected cells were incubated without or with IL-2 (50 ng/mL) for 24 hours. Luciferase activity was measured and normalized for transfection efficiency using a β-galactosidase reporter construct. Data presented represent the mean ± SD of 4 separate experiments. (D) Gel-shift assays were performed using the PRE and WCEs from IL-2R reconstituted and transiently transfected COS-7 cells that were incubated without or with IL-2 (50 ng/mL) for 30 minutes. The location of the specific STAT5b/DNA complex is indicated with the solid triangle; a nonspecific band is indicated with the empty triangle. The arrow indicates the position of the supershifted band. Levels of protein expression, determined by immunoblotting with antibody against STAT5b, are shown in the bottom panel for lanes 1 through 6.