Tyrosine phosphorylation of Shp2 and Stat5 is induced by mutant Kit in HS2 leukemic cells. (A-B) Whole-cell lysates from 606HS2 and 931HS2 cells treated for 4 hours with IM (0.75μM), PP1 (4μM), or PP2 (4μM) or not treated (−; DMSO) were subjected to immunoblotting with the indicated Abs. (C) 663-4A cells overexpressing Kit^D814Y and 663-8A cells overexpressing Kit^D818Y were treated for 4 hours with IM, PP1, or PP2 or not treated (−; DMSO). Whole-cell extracts were subjected to immunoblotting with the indicated Abs. (D) Cell extracts from 606HS2 and 931HS2 cells treated for 4 hours with IM, PP1, or PP2 or not treated (−; DMSO) were immunoprecipitated with anti-Kit Abs. Immunoprecipitates were analyzed by Western blotting with the indicated Abs. Data are representative of 5 independent experiments. (E) HS1 preleukemic cells (633HS1 and 663HS1) were cultured routinely in the presence of SCF (50 ng/mL; +) or starved of SCF in the presence of serum for 4 hours (=) before being stimulated for 10 minutes with SCF (500 ng/mL; +++). Whole cell extracts were subjected to immunoblotting with the indicated Abs.