Identification of the interaction site of FLT3-ITD and SRC. (A) Overview of the mutations created in FLT3-ITD. (B) HEK293 cells transiently expressing either FLT3-ITD or a mutated FLT3-ITD589/591 were starved for 4 hours in serum-free medium and then lysed (right). A pull-down assay was performed using GST alone or the SH2 domain of SRC fused to GST as indicated. Precipitated FLT3 was visualized by immunoblotting using an FLT3 Ab. To confirm equal loading, the membrane was stripped and reprobed with a GST Ab (left). In parallel, lysates were analyzed by immunoblotting for FLT3 expression and STAT5 activation. The membrane was then stripped and reprobed with STAT5 Ab to confirm equal loading (right). (C) Cells stably transfected with FLT3-WT, FLT3-ITD, or FLT3-TKD were starved for 4 hours before stimulation with FL (100 ng/mL) for the indicated times. The lysates were subjected to immunoblotting and the membranes were probed with pY589 and pY591, pan-pY (4G10), and total FLT3 Abs, as described previously.²⁶