Lnk binds to FLT3-WT and FLT3-ITD through the SH2 domain. 293T cells were co-transfected with FLT3-WT/ FLT3-ITD and either empty vector (EV) or various V5-tagged Lnk constructs. Cells were serum starved for 16 hours (−) and treated with FL for 15 minutes (+, when indicated). Lysates were precipitated with either anti-FLT3 (A,B,D) or anti-V5 Abs (C,E,F), and the precipitates were subjected to Western blotting analysis using the indicated Abs. (G) Lysates from either U937 or 32D/FLT3-ITD cells were incubated with GST, GST-Lnk-SH2 (GST-SH2), or GST-Lnk-PH (GST-PH) fusion proteins. After pull-down, protein complexes were analyzed by Western blotting with FLT3 Ab. Equal loading of the GST fusion protein was shown by Coomassie staining of the gel (bottom).