cS5 allows Epo-independent induction of a Stat5-responsive reporter construct but requires tyrosine phosphorylation and DNA-binding for activity. (A) Self renewing WT GFP and cS5-expressing WT and Jak2^−/− erythroblasts were transfected with IL-2R-α-Luc. Before harvesting, cells were stimulated with Epo for 5 hours (+) or left untreated (−). Luciferase expression was measured 12 hours after transfection. Right, schematic representation of the promoter enhancer element from the IL-2R-α gene containing 2 Stat5 response elements (Stat5-RE) fused to the luciferase gene (IL-2R-α-Luc). (B) Schematic representation of the mutants used. 293T cells were transiently transfected with different cS5 constructs, together with a murine EpoR cDNA. Twenty-four hours later, cells were left untreated or treated for 30 minutes with Epo. Extracts were analyzed for tyrosine-phosphorylated Stat5 and total Stat5 by Western blot (C) and DNA-binding of transfected Stat5 constructs by EMSAs on a β-casein-specific promoter sequence (D). Jak2^−/− (E) and EpoR^−/− (F) fetal liver cells were infected with retroviruses encoding GFP, cS5, cS5-EE/AA, or cS5-Y694F and subjected to CFU-E assays. Acid benzidine–positive colonies were scored at day 2.