Proliferation assays and signaling studies comparing dose–response curves and signaling in Ba/F3 cell clones expressing the membrane-tethered or cytoplasmic Mpl fusion protein.

(A) Cell proliferation (MTT) assays of Ba/F3 cell clones expressing either the (i) cytoplasmic or the (ii) membrane-tethered form of Mpl. Proliferation data are represented as a fraction of the proliferative response of Ba/F3 cells to 5% conditioned media containing murine IL-3. (B) JAK2, MAPK, and STAT5 phosphorylation were assessed by Western blot analysis in Ba/F3 cell clones expressing either wild-type Mpl [MPL (WT)] or cytoplasmic [MPL (CYTO)] or membrane-targeted [MPL (MEM)] Mpl fusions. Wild-type Mpl was activated using thrombopoietin, whereas Mpl fusions were activated by adding AP20187 (100 nM) for the time indicated (in minutes). IP, immunoprecipitation; WCL, whole cell lysate.