STAT5b mutations Y665F and N642H increase the transcriptional activity of STAT5. (A) HeLa cells were transfected simultaneously with a wild-type STAT5b vector or STAT5b mutants Y665F or N642H, and a luciferase reporter construct with a STAT5-responsive element. Each condition was tested in triplicate and the fold increase compared with wild-type vector is presented. (B) HeLa cells transfected with empty vector, wild-type STAT5b or mutant construct Y665F or N642H were lysed, and STAT5 and phospho-STAT5 levels were analyzed by western blot. (C) The western blot results shown as a fold change of the intensity of phospho-STAT5 expression compared with total STAT5 protein, normalized to the phospho-STAT5/STAT5 expression levels of HeLa cells with the wild-type STAT5 construct (Odyssey Imaging System; LI-COR Biosciences). wt, wild type; ep, empty plasmid.