Resistance of KIT mutants against apoptotic cell death and hyperphosphorylation of MAPK in response to SCF. (A-B) Ba/F3 cells transduced with the indicated constructs (A: •, KIT-WT; ○, T417I/Δ418_419; ▾, D816V) were grown for 48 hours in the presence or absence of SCF as indicated (B: ▪, no factor; , 1 ng/mL; , 10 ng/mL; □, 100 ng/mL) and subsequently analyzed by flow cytometry after staining with annexin V-PE and 7-AAD. All values obtained from D816V and T417I/Δ418_419 cell lines and all values of Δ419 and Δ418_419 labeled with an asterisk were significantly different from the corresponding wild-type value (P < .05). Figures show mean values and SDs from 3 independent experiments. Data were statistically tested using a 2-sided paired t test (Excel, Microsoft). (C) Ba/F3 cells transduced with the indicated constructs were starved for 24 hours in the presence of 0.3% fetal bovine serum (FBS) and stimulated with the indicated concentrations of SCF or IL-3 (50 ng/mL) for 5 minutes at 37°C and 5% CO₂. Crude lysates were analyzed by Western blotting using a polyclonal phospho-specific mitogen-activated protein kinase antibody (α pMAPK). Equal expression of MAPK in the same lysates was determined by immunoblotting with a polyclonal MAPK antibody (α MAPK); only the reblot for the 10 ng/mL dose of SCF is shown.