EPO signaling mediates phosphorylation of JAK2 and the EPOR but not STAT-5 in HT-2 cells. (A) HT-2.EPOR.3 cells were rested and stimulated with no cytokine (U), 50 U/mL EPO (E), or 10 nmol/L IL-2 (2) and lysates were immunoprecipitated with 5 μg of antibodies to JAK1 (lanes 1 through 3), JAK2 (lanes 4 through 6), or JAK3 (lanes 7 through 9). Immunoprecipitates were separated on 8.75% SDS-PAGE, transferred to nitrocellulose, and probed with antiphosphotyrosine antibodies. Migration of molecular-weight markers is indicated. The presence of a small amount of phosphorylated JAK3 in lanes 7 and 8 is probably background phosphorylation resulting from incomplete IL-2 deprivation of the cells before restimulation with the indicated cytokines. (B) HT-2.EPOR.3 and 371.2 cells were rested and stimulated for 15 minutes with no cytokine (U), 50 U/mL EPO (E), 10 nmol/L IL-2 (2), or 10% WEHI-CM (IL-3) (3) and nuclear extracts were prepared. EMSAs were performed with 10 μg of nuclear extracts and a³²P-labeled oligonucleotide corresponding to the FcγRI STAT-response element.46 Supershifts were performed by preincubating nuclear extracts with anti–STAT-5A and -5B antisera (lane 4) or preimmune rabbit sera (lane 5) for 45 minutes before the EMSA reaction. (C) HT-2.EPOR.3 cells were rested and stimulated with no cytokine (U), EPO (E), or IL-2 (2) and lysates were immunoprecipitated with antibodies to STAT-5A and -5B. Immunoprecipitates were separated on 8.75% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antiphosphotyrosine antibodies. The membrane was stripped and reprobed with anti–STAT-5A and -5B antibodies. (D) 5 × 10⁶ HT-2.EPOR.3 cells/sample or 2 × 10⁶371.2.EPOR cells/sample were rested and incubated with no cytokine (U) or 50 U/mL EPO (E) and lysates were immunoprecipitated with anti-EPOR antibodies. Immunoprecipitates were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with antiphosphotyrosine antibodies.