Lyn induces tyrosine phosphorylation of Stat5 and activates its DNA-binding ability in COS7 cells. (A) The EpoR and ovine Stat5 tagged with an epitope recognized with anti-HA were transiently coexpressed with Jak2 or with both LynA and LynB, as indicated, in COS7 cells. Cells were left unstimulated (−) or stimulated (+) with Epo for 5 minutes before solubilization, as indicated. Cell lysates were subjected to immunoprecipitation with anti-Stat5A followed by immunoblotting with the indicated antibodies. (B) The EpoR and wild-type ovine Stat5 (W) or its mutant with a substitution of Tyr694 with Phe (M) were coexpressed in COS7 cells with Jak2 or Lyn, as indicated. Cell lysates were immunoprecipitated with anti-Stat5A and subjected to immunoblotting with indicated antibodies. (C) The EpoR and ovine Stat5 were coexpressed with Jak2 or Lyn in COS7 cells, as indicated. Cells were left unstimulated (−) or stimulated (+) with Epo for 15 minutes before solubilization, as indicated. Cell lysates were subjected to affinity purification with a PIE oligonucleotide. Total cell lysates (TCL) or affinity-purified proteins (PIE) were analyzed by immunoblotting with indicated antibodies.