Differential activation of signaling pathways by the Flt3 mutation classes. (A) Differential STAT5 activation. The 32D cells stably expressing the indicated Flt3 constructs were starved overnight in medium containing 0.5% FCS with or without the addition of FL as indicated. Total cell lysates were separated by SDS-PAGE. After blotting, the blots were stained using an activation state specific phospho-STAT5 (Y694/699) antibody. Subsequently, the membrane was stripped and reprobed using an anti-CIS antibody, as indicated. Equal loading was shown by reprobing the membrane with an antibody recognizing total STAT5. For analysis of the Pim-2 expression levels, the proteins were resolved on a 15% gel and stained with a Pim-2 antibody. Equal loading was confirmed by reprobing the blot with an antibody specific for β-actin. (B) The transcription factors Pu.1 and C/EBP-α are specifically repressed by Flt3-ITD mutations. Cells were grown for 36 hours in the absence of IL-3 and with (Flt3-WT) or without (Flt3-D835Y and Flt3-ITD) FL in medium supplemented with 10% FCS. The lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for c/EBP-α or Pu.1, as indicated. Membranes were stripped off and reprobed for β-actin to ensure equal loading. (C) Differential activation of STAT5, but not of Erk, by ITD versus D835Y in primary AML samples. Cell lysates were prepared from frozen bone marrow and total cell lysates were analyzed for activation of MAP kinase or STAT5 using activation state-specific antibodies, as described. (D) Preferential activation of STAT5 by Flt3-ITD in AML samples. The activation of STAT5 was analyzed as described in panel C. P indicates patient.