TPO-induced signaling in WT and Tg(mpl) megakaryocytes. Bone marrow cells were harvested from mice and grown in serum-free medium with 37.5 ng/mL mTPO for 3 days; megakaryocytes were then purified using an albumin density-gradient column achieving a final enrichment of greater than 90%. (A) Western blots using Mpl antiserum demonstrates the relative Mpl expression in both WT and Tg(mpl) megakaryocytes. (B) Isolated megakaryocytes were incubated in a serum-free, cytokine-free medium for 10 hours and then stimulated with 50 ng/mL or 500 ng/mL mTpo for 12 minutes. Total cell lysates, normalized for protein concentration, were separated by 4% to 20% SDS-PAGE and immunoblotted with the indicated phosphospecific antibodies (Jak2, Stat3, Stat5, and MAPK; “Methods”). Blots were then stripped and reprobed with appropriate antibodies to ensure equal amounts of protein in each lane (bottom panels). Relative phosphorylation levels were quantitated by densitometry. Results are representative of 2 independent experiments.