No detectable effect of overexpression of Ubc5 on activation of Stat5A and expression of Stat5 target gene. (A) Overexpression of Ubc5b in 32D (EpoR wt) cells. Cell lysates from parental 32D (EpoR wt) cells (parental) and 32D (EpoR wt) cells overexpressing human Ubc5b (Ubc5b) were subjected to direct Western blot analyses with anti-Ubc5b antibodies. (B) No effect of Ubc5b overexpression on activation of Stat5A. Parental 32D (EpoR wt) cells or 32D (EpoR wt) cells overexpressing human Ubc5b were starved overnight, followed by stimulation with IL-3. In the continuous presence of IL-3, cytoplasmic and nuclear extracts were prepared at the indicated time points and subjected to Western blot analyses with anti–phospho-Stat5 (α-pStat5), anti-Stat5A (α-Stat5A), or anti–nuclear protein YY1 (α-YY1) antibodies. (C) No effect of Ubc5b overexpression on down-regulation of Stat5A in the cytoplasm or the nucleus. Parental 32D (EpoR wt) cells or 32D (EpoR wt) cells overexpressing human Ubc5b were starved overnight, followed by stimulation with IL-3. After removal of the cytokine, cytoplasmic and nuclear extracts were prepared at the indicated time points and subjected to Western blot analyses with anti–phospho-Stat5 (α-pStat5), anti-Stat5A (α-Stat5A), or anti–nuclear protein YY1 (α-YY1) antibodies. (D) No effect of Ubc5b overexpression on expression of Stat5 target gene. Parental 32D (EpoR wt) cells or 32D (EpoR wt) cells overexpressing human Ubc5b were starved overnight, followed by stimulation with IL-3. At the indicated time points, total RNA and subsequent cDNA were prepared. Serial dilutions of the cDNA were subjected to PCR using primers designed to detect the Stat5 target gene, Cis. The β-actin gene was used as an internal RNA level control.