The activation-specific STAT5 antibody specifically immunoprecipitates EPO-R.

(A) Selected EPO-R tyrosine to phenylalanine mutants were generated. The nomenclature is indicated for the wild-type (wt) EPO-R. (B) 293T cells were transiently transfected with the indicated plasmids as shown. Twenty-four hours after transfection, cells were depleted of serum for 18 hours and then stimulated in the presence or absence of 50 U/mL EPO for 10 minutes. Lysates were prepared, and immunoprecipitations (IP) were performed with the activation-specific STAT5 antibody. Tyrosine-phosphorylated proteins were detected by immunoblotting with the monoclonal 4G10 antiphosphotyrosine antibody. The mobility of STAT5 and EPO-R are indicated.