Somatic fusions between HT-2.EPOR.3 and Ba/F3 cells restore EPO-dependent signaling. (A) Indicated cell lines were incubated without cytokine (U), 50 U/mL EPO or 10% WEHI-CM (as a source of IL-3), and [³H]thymidine incorporation measured after 48 hours. (▪), HB40; (▨), HB55; (▤), Ba/F3; (□), HT-2.EPOR.3. (B) Indicated cell lines were incubated without cytokines or with various doses of EPO, and [³H]thymidine incorporation was measured after 48 hours. (▪), HB40; (•), HB55. (C) HT-2.EPOR.3, HB40, and HB55 cells were rested and stimulated for 15 minutes with no cytokine (U), 50 U/mL EPO (E), or 10 nmol/L IL-2 (2). Nuclear extracts were subjected to EMSA with the FcγRI probe. Note that all lanes were derived from the same gel, but in the photograph, irrelevant intervening lanes were omitted between lanes 3 and 4. (D) HT-2.EPOR.3, HB40, and HB55 cells were rested and stimulated with no cytokine (U), EPO (E), or IL-2 (2) and lysates prepared and immunoprecipitated with anti-STAT-5A and -5B antibodies. Lysates were separated on 8.75% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antiphosphotyrosine antibodies. Membranes were stripped and reprobed with anti–STAT-5A and -5B antibodies to confirm equivalent loading. (E) HT-2.EPOR.3, HB40, and HB55 cells were starved and stimulated with no cytokine (lanes 1, 5, and 9) or 50 U/mL EPO (lanes 2 through 4, 6 through 8, and 10 through 12) as described in the legend to Fig 5. RNA was separated on a denaturing agarose/formeldehyde gel, transferred to Zeta Probe membranes, and probed with ³²P-labeled c-fos orGAPD cDNA probes.