The activation-specific STAT5 antibody selectively recognizes the STAT5 docking site on the EPO-R.

(A) Selected EPO-R tyrosine to phenylalanine mutants were generated. The nomenclature is indicated for the wild-type EPO-R. (B) Cell lines expressing each EPO-R construct were incubated for 4 hours in the absence of cytokine and then stimulated with 50 U/mL human EPO for 10 minutes. An immunoprecipitation (IP) was performed with a peptide-specific EPO-R antibody. The extent of tyrosine phosphorylation was detected by Western blotting using the monoclonal antiphosphotyrosine antibody, 4G10. The mobility of the EPO-R is indicated. (C) One hundred micrograms of lysate was resolved via SDS-PAGE, and a Western blot was performed using a peptide-specific EPO-R antibody. The mobility of the EPO-R is indicated. (D) The identical cell lines were incubated for 4 hours in the absence of cytokine and then stimulated with 50 U/mL human EPO for 10 minutes. Lysates were prepared under denaturing conditions, and immunoprecipitations were performed with an activation-specific STAT5 antibody. A Western blot was then performed with the monoclonal antiphosphotyrosine antibody, 4G10 (top). The membrane was stripped and reprobed with a peptide-specific STAT5 antibody (bottom). The mobility of STAT5 and EPO-R are indicated.