Fig. 8.

SHIP2 is required for BCR–ABL-mediated phosphorylation of SHC1. a, b Wild-type (wt) 293T cells and cells with SHIP2 deleted by CRISPR/Cas9 (SHIP2^Crispr) were transfected by BCR–ABL, and analyzed for presence and phosphorylation (p) of given proteins by western blot. In contrast to wt cells, BCR–ABL could not phosphorylate SHC1 in SHIP2^Crispr cells. Addition of wt SHIP2 into the SHIP2^Crispr cells fully rescued BCR–ABL-mediated SHC1 phosphorylation, while the addition of catalytically inactive (phosphatase-dead, PD) had only minor effect. Same results were obtained with two independent SHIP2^Crispr cell lines (not shown). b Quantification of SHC1 phosphorylation in (a), normalized to BCR–ABL levels. Bars represent averages from six experiments (n) with indicated SD, statistically significant differences are indicated (Student’s t test, ***p < 0.001). c SHC1 interacts with BCR–ABL in SHIP2^Crispr 293T cells. BCR–ABL was transfected to wt and two clones of SHIP2^Crispr 293T cells (1KF3, 3KF6), and immunoprecipitated (IP). SHC1 co-immunoprecipitates with BCR–ABL in SHIP2^Crispr, suggesting that lack of SHC1 phosphorylation in SHIP2^Crispr cells is not due to loss of interaction with BCR–ABL. Data are representative for 3 independent experiments (n). d K562, LAMA-84 and KYO-1 cells were seeded at 500 cells/μl, and treated with AS1949490 or DMSO. Cells were counted 48 and 96 h after treatment. Data represent averages ± SD from three independent experiments (n), with two technical replicates for each treatment. Statistically significant differences are indicated (Student’s t test, *p < 0.05, ***p < 0.001)