Constitutive activation of MAP kinase, Akt, and Shc by Flt3-ITD and Flt3-D835Y. (A) Constitutive activation of mitogen-activated protein (MAP) kinase Akt and Shc by Flt3 mutants. The 32D cells stably expressing the indicated Flt3 constructs were starved overnight in medium containing 0.5% FCS with or without the addition of FL as indicated. Total cell lysates were separated by SDS-PAGE. After blotting, the blots were stained with the indicated activation-specific antibodies. phospho-Erk recognizes Erk-1 (p44), and Erk-2 (p42), phosphorylated on Tyr204; phospho-Akt is specific for Akt phosphorylated on S473 and phospho-Shc recognizes Shc phosphorylated on Y239/240. Subsequently, the blots were stripped and stained with the indicated antibodies recognizing above proteins in their nonactivated state to demonstrate equal loading. (B) The activation of Shc, Erk, and Akt was quantitated by densitometry from Western blots (from panel A).