PKC412 inhibits receptor phosphorylation and the constitutive activation of downstream signaling cascades in Ba/F3-FLT3-N841I cells. FLT3-N841I–expressing Ba/F3 cells cultured in either the absence or presence of IL-3 were treated with PKC412 or STI571 (in the absence of IL-3) at the concentrations indicated and lysed in NP-40 buffer after 15- and 30-minute treatments. (A) Whole-cell lysates were subjected to direct immunoblot analysis (top panel) or were used as the starting material for anti-FLT3 immunoprecipitation (bottom panel). Whole-cell extracts or immunoprecipitates were immunoblotted with either anti-pTyr antibody or anti-FLT3 (as indicated). (B) The same cell lysates were subjected to immunoblot analysis with antibodies specific to the phosphorylated forms of STAT5 and ERK, as indicated. Membranes were then reprobed with the corresponding nonphosphospecific antibodies, as indicated.