Repression of STAT5-dependent transcription by SHD1. (A) 293T cells were transfected with plasmids expressing Epo receptor, SHD1, and the luciferase reporter construct containing 4 tandem repeats of optimal STAT5-binding site. One microgram of each plasmid was used for transfection unless otherwise indicated. The cells were stimulated with recombinant human Epo (25 U/mL) after 24 hours of transfection, and harvested for luciferase assay after 24 hours of stimulation. The data are the mean plus or minus SD (n = 3). The lysates were analyzed by Western blotting to determine the expression of STAT5 and α-tubulin as a loading control. (B) The indicated amounts of SHD1 or STAT5A expression vector together with luciferase reporter construct containing β-casein promoter were transfected into BaF3 cells by DEAE-dextran method. The cells were stimulated with recombinant murine IL-3 after 24 hours of transfection and harvested for the analysis after 24 hours of stimulation. The data are the mean plus or minus SD (n = 3). The expression level of STAT5 was analyzed by immunoprecipitation and Western blotting. (C) A vector expressing full-length STAT5B fused to GAL4 DNA-binding domain (pM/STAT5, 1 μg) was transfected into 293T cells together with indicated amounts of SHD1 expression vector and the luciferase reporter plasmid bearing 5 copies of GAL4-binding sites (GAL4-luc). The cells were lysed for the analysis after 48 hours of transfection. The data are the mean plus or minus SD (n = 3).