Analysis of the MPLY591N mutant. (A) MPL cell-surface localization in Ba/F3 cells. Ba/F3 cells expressing the bicistronic retroviral pMEGIX-IRES-GFP vector encoding HA-tagged MPLWT or MPLY591N and GFP were maintained in IL3-supplemented medium. Flow cytometry analysis was assessed and histograms show equivalent GFP expression (left) monitoring the total MPL levels and HA-PE labeling (right) corresponding to cell-surface expression of MPLWT and MPLY591N. (B) Sensitivity to TPO of Ba/F3 cells. Ba/F3-MPLWT or MPLY591N cells were cultured for 72 hours either in absence of cytokine (black arrow, x-axis) or in presence of increasing doses of TPO (0.01, 0.1, 1, and 10 ng/mL). Viable cells were quantified by WST-1 proliferation assay. Dose-response curves are means expressed in percentages of maximum growth value ± SEM (n = 3 in triplicate). Two-tailed t test; *< .05; **P < .01. (C) Signaling studies in Ba/F3-MPL cells. Ba/F3-MPLWT or MPLY591N cells were serum- and cytokine-starved for 6 hours prior to a 15-minute stimulation without or with TPO (0.01, 0.1, 1, and 10 ng/mL) 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 antiphospho-specific antibodies, as indicated. MPL expression was also verified. Expression of β-actin 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 3 independent experiments.