Constitutive activation of STAT5A and 5B in K562 cells. (A) K562 cells (3.10⁶ per lane) and U937 cells (negative control) were lysed in RIPA buffer, after which the tyrosine phosphorylation state of different STAT molecules was assessed by immunoprecipitation and Western blotting. Probing the blot with an antiphosphotyrosine antibody clearly shows that only STAT5A and STAT5B are phosphorylated on tyrosine in K562 cells. (B) Nuclear extracts from K562 cells were analyzed in a gel shift assay using the STAT binding site from the β-casein promoter as a probe. Supershift analysis clearly shows that STAT5A and 5B constitutively bind to the β-casein site in K562 cells.