SSA is regulated by BCR-ABL through the PI3K and Ras/MEK pathways. (A) BaF3 cells containing BCR-ABL or BCR-ABL.Y177F were used. Cells were cotransfected with SAGFP and I-SceI expression vector and assayed for SSA frequency by flow cytometry (n = 5). (B) Phosphorylated Y177 in the active BCR-ABL oncoprotein provides a docking site for Grb2. Signaling downstream of Y177 includes activation of PI3K, Akt, and Ras/MEK pathways. Pharmacologic inhibition of PI3K with LY294002, Akt with Akt inhibitor X, MEK1 with PD98059, or MEK1/2 with U0126 blocks these pathways. (C) I-SceI was expressed in BaF3.BCR-ABL cells with integrated SAGFP and treated for 48 hours with LY294002 (n = 4), PD98059 (n = 4), U0126 (n = 4), or Akt inhibitor X (n = 3). (D) I-SceI was expressed in BaF3.BCR-ABL cells with integrated SAGFP and transfected with control siRNA, siRNA targeting Akt1/2, or MEK1 (n = 3). (E) Cellular expression of HA-tagged I-SceI (HA), actin, Akt1/2/3, or MEK1 was detected by immunoblotting in response to inhibitors or siRNA, as indicated.