Constitutive activation of FLT3 signaling by the FLT3 N676K mutant. Ba/F3 cells expressing indicated constructs were starved for 24 hours in media containing 0.3% fetal calf serum. Cells were left untreated or were stimulated with 100 ng/mL FL for 10 minutes. Crude cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed by western blot for phosphorylation of signaling molecules. (A) STAT5, AKT, and MAPK activation was analyzed by using phospho-specific antibodies, and then stripped and reprobed with antibodies against total STAT5, AKT, and MAPK. Ba/F3 native cells were stimulated with 100 ng/mL IL-3 for 5 minutes; control and an antibody against GAPDH were used as loading control. (B) FLT3 receptor was immunoprecipitated with polyclonal FLT3 antibody, analyzed for tyrosine phosphorylation status by immunoblotting with a phospho-tyrosin antibody, stripped, and reprobed with FLT3 antibody.