The STAT5A/WKR, STAT5A/VVV, and STAT5A/▵53C mutants exert dominant negative effects on erythropoietin-induced, STAT5-dependent transcription. The indicated STAT5A mutant in the expression vector pcDNA3 ([▪] 1 μg; [▧] 2 μg) was cotransfected into 293T cells together with wild-type STAT5A plasmid (0.25 μg), a murine erythropoietin receptor expression construct, β-casein luciferase reporter, and hGH expression plasmid as an internal control. Cells were stimulated with erythropoietin as described in Materials and Methods, and the increase in luciferase activity relative to unstimulated cells, normalized for transfection efficiency, is shown. Omission of the erythropoietin receptor expression construct from the transfection resulted in background levels of erythropoietin-stimulated luciferase activity in transfected cells (data not shown). pcDNA3 indicates cotransfection with vector alone. Error bars represent the standard error based on at least two independent experiments.