Stat5a mutants exhibit unchanged biochemical properties. (A) Schematic representation of mutant Stat5a proteins. The oncogenic Stat5a mutant cS5 contains a Ser to Phe mutation at position 710, which renders it constitutively active. The Stat5a mutants S725A, S779A, and SASA carry single or double Ser to Ala substitutions. (B) 293T cells were transfected with the WT (S5) and hyperactive (cS5) mutant Stat5a constructs indicated and stimulated via the erythropoietin (Epo) receptor. Cells were treated (+) with 50 U of Epo or left untreated (−). Whole-cell extracts were prepared and subjected to Western blot analysis to evaluate tyrosine phosphorylation. (C) Epo-stimulated extracts from panel B were subjected to EMSA using the β-casein site to study the DNA binding properties of the different WT Stat5a and hyperactive Stat5a mutants. Equal dimer activity was established before analyzing the extracts in tetramer assays as described previously.^2,28  (D) Tetramer formation of the Stat5a mutants indicated was analyzed with Epo-stimulated 293T cell extracts from panel B using a 2× β-casein binding element. All Stat5a derivatives were found to bind to the site as tetramers. Compared with dimers, tetramers were more stable toward competition by cold DNA. The absence of cold DNA (0) represents the saturated binding reaction. This was followed by a 100× competition by cold DNA for 5, 10, and 25 minutes. cS5, cS5-S725A, and cS5-S779A form tetramers with enhanced DNA binding activity, which was in contrast to WT Stat5a (S5) and cS5-SASA. The data are representative for 3 individual transfections.