Lyn regulates Gab2 tyrosine phosphorylation, BCR-ABL signaling complexes, and imatinib activity in CML cells. (A) Gab2 was immunoprecipitated from K562 and K562R equal protein (1 mg) cell lysates and immunoblotted for phosphotyrosine (pY-Gab2, pY-BCR-ABL), BCR-ABL, Gab2, and Lyn. (B) K562R cells were treated with imatinib or dasatinib at the indicated concentration for 2 hours prior to Gab2 immunoprecipitation from equal protein cell lysates (0.5 mg) and blotting for phosphotyrosine (top) or Gab2 (bottom). Cell lysates subjected to immunoprecipitation media in the absence of anti-Gab2 were used to detect any nonspecific proteins (last lane). (C) K562R cells were electroporated with the indicated siRNA for 48 hours before immunoprecipitation of Gab2 and blotting for phosphotyrosine content and Gab2 levels. Lyn levels were measured by immunoblotting cell lysate. SHP1 was immunoblotted and used as a protein loading control. (D) K562R cells were electroporated with control or Gab2 siRNA (100 nM) and cell death was assessed by trypan blue exclusion (below) after 48 hours. Cell lysates were also examined for Gab2 protein levels by immunoblotting. Actin was probed as a protein loading control. (E) K562 or K562R cells were subjected to electroporation with 100 nM of either control or Lyn siRNA as described in “Methods.” After 24 hours, cells were treated with 2.5 μM imatinib (+) or vehicle alone (−) for an additional 2 hours before analysis of BCR-ABL, Lyn, and Y177-BCR-ABL levels by immunoblotting. (F) K562R cells were electroporated with 100 nM control, Hck, or Lyn siRNA (as indicated). After 24 hours, electroporated cells were treated with vehicle alone (none) or 2.5 μM imatinib for an additional 24 hours. Cells were also treated with dasatinib alone (0.2 μM) (without electroporation) for 24 hours before all cells were collected and cell death was estimated by trypan blue staining. The results represent the average plus or minus SEM of triplicate assays.