Lyn regulates BCR-ABL tyrosine phosphorylation in CML cells. (A) Cos-7 cells were transfected with a kinase-inactive mutant of BCR-ABL (K271R) or cotransfected with kinase-active Lyn. After 24 hours, cotransfectants were treated with the indicated concentration of kinase inhibitor for 2 hours. Cell lysates were prepared and immunoblotted for site-specific (Y177) BCR-ABL phosphorylation, BCR-ABL, Lyn, and actin levels. Low-level Y177-BCR-ABL was detected in BCR-ABL (K271R)–transfected Cos-7 cells (lane 2) that were not affected by imatinib or dasatinib. The vertical cut line between lanes 2 and 3 denotes merging of the original image to eliminate experimentally irrelevant sample lanes. (B) A leukopheresis specimen from a CML lymphoid blast crisis patient who progressed on imatinib therapy was left untreated (control) or was treated with imatinib (5 μM) or dasatinib (0.25 μM) for 2 hours. Equal protein cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted for the antigen described. For detection of tyrosine-phosphorylated Lyn, 1 mg protein lysate from control or treated cells was subjected to Lyn immunoprecipitation and phosphotyrosine immunoblotting (pY-Lyn). The blot was stripped and reblotted for Lyn (bottom). (C) Specimens from 4 imatinib-resistant myeloid blast crisis patients (Res1-Res 4) that retained wild-type BCR-ABL expression were treated with imatinib or dasatinib (at the concentration indicated) for 2 hours. Equal protein cell lysates were subjected to immunoblotting for phospho-specific and total protein levels (as indicated). K562R cell lysate was used as a positive control for Lyn and other phosphoproteins.