MAPK activation in CB-derived megakaryocytes. (A) Kinetic of ERK activation in day 7 and day 10 cells derived from purified CD34⁺ cells grown in the presence of rhuMGDF. Cells were deprived of growth factor by incubation for 5 hours in cytokine-free medium and stimulated for the indicated times with 100 ng/mL rhuMGDF. (B) Inhibition of rhuMGDF-induced ERK activation by PD98059. Cells were grown for 10 days with rhuMGDF, starved, and stimulated for 60 minutes at 37°C with 100 ng/mL of rhuMGDF in the presence of 6 μmol/L PD98059 (+) or the equivalent amount of DMSO as control (−). ERK activity was detected by immunoblotting whole-cell lysates with antiphospho ERK antibody. The amount of sample loaded in each lane was verified by immunoblotting the same membranes with an antibody recognizing both active and inactive ERK1 and ERK2.