The FLT3 PTK inhibitor SU5614 down-regulates autophosphorylation of the FLT3-ITD but not FLT3-D835H receptor mutants. (A) 293T cells were transiently transfected with empty vector (MIY), FLT3-ITD, and FLT3-D835H constructs. Forty-four hours after transfection the cells were incubated with SU5614 as indicated for 4 hours and lysed. FLT3 was immunoprecipitated with a polyclonal anti-FLT3 antibody. Tyrosine phosphorylation of FLT3 was determined by Western blot analysis with the use of a monoclonal anti-PY antibody, and identical loading was confirmed by reblotting with a polyclonal anti-FLT3 antibody. (B) The expression and phosphorylation of STAT5 in Ba/F3 MIY-, FLT3-ITD–, and FLT3-D835H–expressing cells treated with 0.1, 1, and 10 μM SU5614 or untreated cells was determined by Western blot analysis in crude lysates with the use of polyclonal pSTAT5 and anti-STAT5 antibodies.