Lyn phosphorylates the EpoR and Stat5 on tyrosine in vitro. 32D/EpoR-Wt cells were left unstimulated or stimulated with Epo, as indicated. Cells were then lysed and immunoprecipitated with anti-Lyn (Lyn +) or with normal rabbit serum (Lyn −). GST-EpoR (upper panels) and GST-Stat5 (lower panels) proteins were then added to immunoprecipitates as substrates and incubated at room temperature for 30 minutes in in vitro kinase buffer with or without 1 mmol/L ATP, as indicated. Reaction products were then subjected to immunoblotting with antiphosphotyrosine followed by reprobing with anti-EpoR or anti-Stat5, as indicated.