Fig. 3. Effects of SMRT on transcriptional activity of STAT5. (A) Full-length STAT5B fused to the GAL4 DNA-binding domain (GAL4–STAT5BF) and SMRT were co-transfected into Y190, which bears the LacZ reporter driven by GAL4. SMRT expression was induced by addition of CuSO₄ and the β-gal activities induced by GAL4–STAT5BF were compared. β-gal activity was measured according to the protocol described in Materials and methods. (B) GAL4–STAT5B or GAL4-VP-16 and SMRT were co-transfected into 293 cells with luciferase reporter plasmid bearing five copies of the GAL4-binding site (GAL4-Luc). Cells were harvested and lysed after 48 h of transfection. Luciferase activity of the extract was examined by luciferase assay systems (Promega). Data were normalized according to the β-gal activity by co-transfected RSV-β-gal plasmid. (C) 293 cells were transfected with a reporter plasmid bearing four copies of optimal STAT5 binding site (ST5BS-Luc), Epo receptor (EpoR), SMRT and wild-type STAT5 or STAT5ΔC. Cells were treated with or without 25 U/ml recombinant human Epo after 24 h of transfection. After 24 h of Epo stimulation, cells were harvested and lysed for luciferase assay. Data are shown as fold activation. (D) Luciferase assay using β-casein promoter. EpoR, STAT5, SMRT and β-casein reporter construct were transfected into 293 cells. Epo stimulation and cell lysis were performed similarly as in (C). Data are shown as relative luciferase activity. (E) Effects of TSA on the repressive effect of SMRT. EpoR, SMRT and ST5BS-Luc construct were transfected into 293 cells. Cells were treated with 100 ng/ml TSA together with 25 U/ml Epo for 24 h before cell harvest. Data are shown as fold activation. (F) Effects of SMRT on transcription by various STATs. Full-length STAT1, 4, 5A, 5B and 6 were fused to the GAL4 DNA-binding domain and transfected into 293 cells with SMRT and GAL4-Luc reporter. Repressive effects of SMRT are shown as fold repression.