Sustained binding of Stat5-Y694 to tyrosine-phosphorylated β_IL3 and its inhibitory effect on tyrosine phosphorylation of endogenous Stat5. A clone of BaF3 cells expressing wild-type ovine Stat5 (Stat5-Wt) or mutant ovine Stat5 in which Tyr694 was changed to Phe (Stat5-Y694F ) was stimulated with 25 ng/mL of IL-3 for the indicated times. Cells were then lysed and subjected to immunoprecipitation with anti-HA (A), anti-Stat5A (B), or anti-Stat5B (C). Immunoprecipitates were subjected to immunoblotting with the antibody indicated (upper panels) and then reprobed with the other antibodies indicated (middle and lower panels).