Epo induces tyrosine phosphorylation of β_IL3 in 32D/EpoR-Wt cells. In (A) and (B), 32D/EpoR-Wt cells, a clone of IL-3–dependent 32D cell line expressing the transfected wild-type murine EpoR cDNA, were washed out of IL-3 for 12 hours and left unstimulated (C) or stimulated with 100 U/mL of Epo or 25 ng/mL of IL-3 for 1 or 5 minutes as indicated, at 37°C before solubilization. In (C), 32D/EpoR-Wt cells were stimulated with the indicated concentrations of Epo or 25 ng/mL of IL-3, as indicated, for 1 minute. The cell lysates were immunoprecipitated with anti-β_IL3 (A, C) or anti-EpoR (B). Immunoprecipitates were resolved by 6% SDS-PAGE and subjected to immunoblotting with an antiphosphotyrosine monoclonal antibody, 4G10 (αPY). The membranes were then stripped and reprobed with the antibody used for immunoprecipitation, as indicated.