SRC physically interacts with FLT3-ITD but not FLT3-TKD. (A) HEK293 cells expressing FLAG-tagged FLT3-ITD or FLT3-TKD were serum starved for 4 hours, followed by lysis and pull-down with a GST fusion protein containing the SH2 domain of FYN, LCK, HCK, and SRC. Samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-FLAG Ab. (B) GST pull-down experiments were performed as described in panel A using HEK293 cells transiently transfected with FLT3-WT or the indicated mutants and GST SRC SH2 as bait. Cells were starved for 4 hours and stimulated with FL for 10 minutes or left untreated before lysis. Interaction was analyzed by immunoblotting with a FLAG Ab. FLT3 immunoprecipitation followed by immunoblotting with pY and FLAG Ab was carried out to confirm the phosphorylation status of the expressed proteins. (C) HEK293 cells transiently expressing FLAG-tagged FLT3-WT, FLT3-ITD, or FLT3-TKD were serum starved for 4 hours. FLT3-WT–expressing cells were either untreated or stimulated with FL. FLT3 was immunoprecipitated from whole-cell lysates using a FLAG Ab and immunoblotted with a SRC Ab, stripped, and reprobed with anti-FLAG Ab to confirm equal precipitation. In parallel, an aliquot of each cell lysate was immunoblotted and probed with an Ab against pY and FLAG to verify the proper phosphorylation and expression of the proteins in the cells.