The FLT3-Y842C mutant is constitutively tyrosine phosphorylated and inhibits apoptotic cell death upon growth factor withdrawal. 32D cells were transfected with either pAL-FLT3-WT, pAL-FLT3-ITD, or pAL-FLT3-Y842C. (A) 32D-FLT3-Y842C cells were grown in the absence of IL-3 and incubated with PKC412 (100 nM), FL (100 ng/mL), 10% Wehi-conditioned medium, or in combinations as indicated. MTT assays were performed at several time points. Shown is 1 representative of 2 experiments. (B) 32D-FLT3-Y842C and FLT3-WT cells were treated for 48 hours as indicated and cell cycle analysis was performed. Shown is 1 representative of 2 experiments. (C) Analysis of FLT3 membrane expression by flow cytometry. 32D cells transfected with FLT3-WT, FLT3-ITD, and FLT3-Y842C were stimulated with FL (100 ng/mL) for the time points indicated. FLT3 receptor expression was determined by flow cytometry using a specific PE-conjugated anti-FLT3 antibody. (D) FLT3-Y842C mutants were treated with FL (100 ng/mL), PKC 412 (100 nM), and in combination for 15 minutes as indicated. Whole-cell lysates were prepared and analyzed by Western blot (IB). To control equal loading, the blot was stripped and reprobed with antibodies as indicated. In addition, immunoprecipitation (IP) using a specific anti-Flt3 antibody followed by Western blot analysis with an antiphosphotyrosine (p-Tyr) antibody was performed. IgG indicates immunoglobulin G. ^*Error bar indicates standard deviation.