FLT3 activation loop mutations constitutively activate FLT3 and its downstream effectors, STAT5 and ERK. Cell lysates of mutants were prepared from Ba/F3 cells, grown in the absence of IL-3, stably expressing FLT3-ITD or FLT3 activation loop mutations. Negative control cell lysates were prepared from untransfected Ba/F3 cells starved of IL-3 overnight. Immunoprecipitation (IP) with anti-FLT3 antibody was performed, resolved by SDS-PAGE, and immunoblotting was performed with antiphosphotyrosine antibody or anti-FLT3 antibody, as indicated. Whole cell lysates (WCL) were also resolved by SDS-PAGE, then immunoblotted with anti-phospho-STAT5, anti-phospho-ERK, anti-STAT5, or anti-ERK antibodies, as indicated.