TPO-induced activation of Src kinases. Parental BaF3 cells and those engineered to express c-Mpl were starved overnight. Half of the cells were stimulated with TPO (15 ng/mL) for 10 minutes before making whole-cell lysates. (A) Kinase assays were performed using γ-³²P] ATP and peptides that function as efficient ([Lys¹⁹] cdc2(6-20)-NH₂) and inefficient ([Lys¹⁹Ser¹⁴Val¹²] cdc2(6-20)-NH₂) substrates for Src kinases. (B) Immune complex kinase assay. Lysates normalized for total protein concentration were immunoprecipitated with either anti-Lyn or anti-Fyn antibodies. Kinase activity was measured by phosphorylation of substrate ([Lys¹⁹] cdc2(6-20)-NH₂) in immune complexes. (C) Cells were pretreated for 45 minutes with DMSO (vehicle = 0) or the stated concentration of PP2 and stimulated with TPO. Lysates were immunoprecipitated with anti-Lyn, after which immune complex kinase assays were performed. The data have been normalized as a percentage of maximal kinase activity induced by TPO in the presence of DMSO alone. Immunoblots of immunoprecipitated lysates (A-B) were performed to ensure equal amounts of Lyn and Fyn were assayed (data not shown). All assay data are representative of 3 independent experiments. Error bars indicate SD.