TPO-induced MAPK phosphorylation in BAF3/Mpl cells. BAF3/Mpl cells were cultured in serum-free, cytokine-free media for 16 hours. Twenty minutes before stimulation, PD 98059 (PD) at a final concentration of either 50 or 100 μmol/L, was added. Cells were then stimulated by the addition of 14 ng/mL murine TPO for 10 minutes, lysed, and subjected to Western blot analysis. The blot was probed with anti–double-phosphorylated ERKs/MAPK antibody (upper panel), showing 2 specific bands corresponding to ERK1 (44 kD) and ERK2 (42 kD). ERK phosphorylation could be partially inhibited by PD 98059. The blot was then stripped and reprobed with anti-ERK2 antibody (lower panel) to assure equal loading in all lanes.