Suppression by GM-CSF of EPO-induced erythroid differentiation occurs downstream of the EPO receptor. GM-CSF was removed from UT-7/GM cells overnight. The cells were pretreated with medium alone (lanes 1 and 2) or GM-CSF (10 ng/mL; lanes 3 and 4) for 5 minutes and then stimulated with medium alone (lanes 1 and 3) or EPO (10 U/mL; lanes 2 and 4) for 5 minutes before solubilization. Cell lysates were immunoprecipitated with agarose-conjugated anti-EPOR antibody. Immunoprecipitates were eluted with buffer containing SDS and resolved by 7.5% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and blotted with anti-P-Tyr MoAb (4G10).