(A, upper panel) Tyrosine phosphorylation of STAT3 was not tyrosine phosphorylated in the erythroid cells stimulated with erythropoietin 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). STAT3 was immunoprecipitated with specific anti-STAT3 antisera. As a positive control, TF-1 cells were stimulated with IL-6 (100 ng/mL) for 10 minutes. Immune complexes were resuspended in SDS sample buffer. Tyrosine phosphorylation of STAT3 was detected by 4G10 as described in Fig 1A. Bands were visualized by chemiluminescence. Lane 1, resting erythroid cells; lane 2, 10 minutes after exposure to erythropoietin (10 U/mL); lane 3, resting TF-1 cells, lane 4, 10 minutes after exposure to IL-6 (100 ng/mL). (lower panel) The same nylon membrane was stripped of the antibody and reprobed for STAT3. Bands were visualized by chemiluminescence. Lanes are the same as in upper panel. (B) Anti-STAT1 was used instead of STAT3 antisera.