Activation-specific STAT5 antibodies selectively immunoprecipitate tyrosine-phosphorylated STAT5 and EPO-R.

(A) Ba/F3–EPO-R cells were incubated for 8 hours in the absence of cytokine and then stimulated with 50 U/mL human EPO for 10 minutes. Lysates (Lys) were prepared under nondenaturing conditions (lanes 1-6) or denaturing conditions (lanes 7-12). Immunoprecipitations were performed with the following STAT5 antibodies: total STAT5, activation-specific polyclonal phosphorylated STAT5 (pSTAT5) (David A. Frank [DAF]), and activation-specific polyclonal pSTAT5 (Zymed). A Western blot was performed with the antiphosphotyrosine antibody, 4G10. The mobility of STAT5 and EPO-R are indicated. (B) Splenic erythroblasts were incubated for 4 hours in the absence of cytokine and then stimulated with 50 U/mL human EPO for 10 minutes. Immunoprecipitation (IP) and Western blotting were performed as described above. pTyr indicates phosphotyrosine.