Signaling studies in Ba/F3-EPOR and Ba/F3-MPL cells and STAT activities in γ2A cells. (A) Ba/F3-EPOR or (B) Ba/F3-MPL cells expressing the different JAK2 constructs were serum- and cytokine-starved for 6 hours prior to a 15-minute stimulation with (A) 1 U/mL EPO and (B) 0.05 and 5 ng/mL TPO at 37°C, as indicated. Cells were lysed, and the phosphorylation status of STAT1, STAT3, STAT5, AKT, and ERK1/2 was examined by western blotting with the respective anti-phospho specific antibodies, as indicated. Expression of HSC70 in the samples was used as loading control and was consistent with expression of total AKT, ERK1/2, and the individual STAT isoforms. Blots shown were reproduced in 2 independent experiments. The quantification of the phospho-STAT5 (P-STAT5) blots for unstimulated Ba/F3-EPOR and Ba/F3-MPL were shown as P-STAT5/HSC70 ratios (arbitrary units [AU]) and displayed below the figures. (C) JAK2-deficient γ2A cells were transfected to express the various JAK2 mutants in the presence of equal amounts of JAK2 WT and MPL. STAT1-, STAT3-, or STAT5-dependent transcriptional activity was measured 24 hours after transfection by the dual firefly (pGRR5 reporter responding to STATs) and renilla (pRL-TK reporter with constitutive expression) luciferase system. Shown are averages ± SEM of 9 experiments in triplicate. Two-tailed Student t test: *P < .05; **P < .01; ***P < .001.