Similar signal transduction properties and sensitivity toward PKC412 of Flt3-TKD mutations. (A) Surface expression of Flt3-TKD and ITD mutations. The 32D cells expressing the indicated constructs were analyzed by flow cytometry using an anti–human Flt3 (CD135) antibody. The shaded curves show staining with anti-Flt3 antibody; open curves, the isotype control. (B) Comparison of the DNA synthesis of the different Flt3 mutant cell lines by ³H-thymidine incorporation. 32D cells expressing the indicated Flt3 constructs were starved for 12 hours in medium supplemented with 0.5% serum. Subsequently, cells were exposed to FL or IL-3 or left unstimulated. Data are shown as percentage of thymidine incorporation compared with thymidine incorporation of the respective cell line under IL-3 stimulation. (C) Signaling properties of Flt3 mutants. The indicated cell lines were analyzed for Flt3 autophosphorylation using activation state-specific phospho-Flt3 (Y591) antibody. Flt3 kinase assays were performed as described in “Patients, materials, and methods.” The activation of the STAT5 pathway and expression of myeloid transcription factors was analyzed as described in “Patients, materials, and methods.” (D) The 32D cells containing the indicated Flt3 constructs were analyzed for their proliferation by ³H-thymidine incorporation as described in panel B under increasing concentrations of PKC412. Each data point represents the mean of ³H-thymidine incorporation of 3 samples ± SD. The numbers are given as fraction of DNA synthesis in the absence of PKC412. (E) Effect of FL on sensitivity of Flt3-TKD mutants toward PKC412 was analyzed in presence or absence of FL under increasing concentration of the inhibitor. Data represent mean values ± SD of triplicates.