Fig. 2. Proliferation and high activation of signaling pathways are restored in previously underexpressing clones that were retransduced to increase expression of Mpl. (A) 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. (B) 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. Activation of STAT-5, ERK and AKT were determined as described in Fig. 1. ns, no stimulation.