Inhibition of FLT3 downstream signaling nodes and of FLT3 kinase activity by PKC412 in 32D_ITD627E cells and in 32D_ITD cells. (A) 32D_ITD and 32D_ITD627E cells were treated with or without different concentrations of PKC412 for 2 hours. Protein expression and phosphorylation of FLT3 (Y591), STAT5 (Y694/Y699), AKT (S473), ERK1/2 (T202/Y204), and S6 protein (S240/S244) were determined by immunoblotting. (B) Overall tyrosine phosphorylation of FLT3 in 32D_ITD and 32D_ITD627E cells after treatment with PKC412 for 2 hours was determined by immunoprecipitation of FLT3 and Western blot analysis using anti–phosphotyrosine antibody (4G10). (C) Assessment of in vitro kinase activity of immunoprecipitated FLT3_ITD and FLT3_ITD627E receptors upon incubation in the absence and presence of various concentrations of PKC412. The experiment shown is representative of 4 with consistent results. (D) Overall tyrosine phosphorylation and specific tyrosine phosphorylation of phosphoepitopes of FLT3 in 32D_ITD and 32D_ITD627E cells after treatment with PKC412 for 2 hours was determined by immunoprecipitation of FLT3 and Western blot analysis using anti-FLT3 antibody, anti–phosphotyrosine antibody (4G10), anti-FLT3pY591, anti-FLT3pY589, anti-FLT3pY599, anti-FLT3pY768, anti-FLT3pY955, and anti-FLT3pY969 antibodies. (E) Phosphorylation of ERK1/2 (T202/Y204) was assessed in 32D_ITD627E cells by immunoblotting 24 hours after introduction of FLT3-specific siRNA (left panel). Percentage of apoptotic 32D_ITD627E cells was determined in parallel by flow cytometry 24 hours and 48 hours after siRNA knockdown of FLT3 (right panel).