GRB-2/FLT3_ITD627E interaction is sustained upon dephosphorylation of FLT3 by PKC412. (A) Analysis of GRB-2/FLT3_ITD627E interaction upon incubation with PKC412. FLT3 was immunoprecipitated from protein lysates of 32D_ITD, 32D_ITD627E, and 32D_ITD627A cells after treatment with 10 nM PKC412 for 2 hours, and the amount of coimmunoprecipitated GRB-2 was assessed by immunoblot analysis. As a control, GRB-2 protein expression was determined in lysates (100 μg) from each cell line. (B) Suppression of GRB-2 protein expression by RNA interference rescues sensitivity of FLT3_ITD627E cells to PKC412. The percentage of apoptotic 32D_ITD627E cells was assessed by flow cytometry 48 hours after introduction of GRB-2–specific siRNA and treatment of cells with and without PKC412 (right panel). GRB-2 and MCL-1 expression was controlled at 48 hours by immunoblotting (left panel). Results from 1 representative experiment of a total of 2 are shown.