Inhibition of Lyn alters TPO-induced signaling. BaF3/Mpl cells were starved, pretreated with 2 μm PP2 or DMSO, and were unstimulated (–) or stimulated (+) with TPO 15 ng/mL. Total cell lysates, normalized for protein concentration, were separated by SDS-10% polyacrylamide gel electrophoresis (PAGE) and immunoblotted with specific antibodies. (A) Whole-cell lysates were immunoblotted with a phosphotyrosine antibody (4G10). The numbers on the left indicate the sizes of the molecular mass makers in kilodaltons. (B) Mpl was immunoprecipitated with Mpl antiserum, analyzed by Western blot, and probed with antiphosphotyrosine antibody. (C) JAK2, Shc, STAT3, and STAT5 were immunoprecipitated, subjected to Western blotting, and probed with the indicated phospho-specific antibodies (“Materials and methods”). Arrows to the right of the gel refer to phosphoproteins. Blots were stripped and reprobed with appropriate antibodies to ensure equal amount of protein in each lane.