MLN518 inhibits autophosphorylation of FLT3 and phosphorylation of downstream effectors, STAT5 and ERK, in a pattern similar to that seen for cellular proliferation. (A) MLN518 inhibits FLT3 autophosphorylation. Ba/F3 cells stably expressing FLT3-ITD or FLT3 activation loop mutations were incubated with increasing concentrations of MLN518 for 30 minutes. Cell lysates were prepared, immunoprecipitated (IP) with anti-FLT3 antibody, resolved by SDS-PAGE, then immunoblotted with antiphosphotyrosine antibody or anti-FLT3 antibody, as indicated. (B) Inhibition of downstream STAT5 phosphorylation by MLN518. Whole-cell lysates (WCL) prepared as above were also resolved by SDS-PAGE, then immunoblotted with anti-phospho-STAT5 or anti-STAT5 antibodies, as indicated. (C) Inhibition of downstream ERK phosphorylation by MLN518. Whole-cell lysates prepared as above were also resolved by SDS-PAGE, then immunoblotted with anti-phospho-ERK or anti-ERK antibodies, as indicated.