(upper panel) Tyrosine phosphorylation of STAT5 in the erythroid day-8 cells stimulated by erythropoietin (10 U/mL) for 10 minutes. The erythroid cells were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100 before and after exposure to erythropoietin (10 U/mL). STAT5 was immunoprecipitated with STAT5A or STAT5B antisera as indicated. Immune complexes were resuspended in SDS sample buffer. Tyrosine phosphorylation of STAT5 was detected by 4G10 as described in Fig 1A. Bands were visualized by chemiluminescence. (lower panel) The same nylon membrane was stripped of the antibody and reprobed for STAT5 with an anti-STAT5 monoclonal antibody. Bands were visualized by chemiluminescence.