Fig. 1. A threshold number of cell-surface Mpl receptors is necessary for TPO-induced proliferation of BaF-3 cells. (A) Flow cytometric analysis of Mpl-expressing clones. A Flag-Mpl receptor construct was introduced into BaF-3 cells and clones were derived. Cell surface expression of Mpl was examined for each clone by Flag-PE immunostaining: broken line, unlabeled cells; solid line, labeled cells. (B) Proliferation assay. Cells were incubated with the indicated concentration of TPO for 48 hours and cell proliferation was quantitated by [³H]thymidine incorporation. The results shown are the means±s.d. of triplicate experiments. (C) Western blots analysis of signaling pathways activated in clones stimulated by TPO. Cells were washed three times, deprived of cytokines for 3 hours, and then stimulated with 50 ng/ml of TPO for 15 minutes. Lysates (60 µg of protein per lane) were fractionated by SDS-PAGE.

Phosphorylated (upper panel) and total (lower panel) STAT-5, ERK and AKT were analyzed by immunoblotting. ns, no stimulation. The positions of ERK1 (p44) and ERK2 (p42) are indicated.