Forced expression of Lnk inhibits FLT3-ITD–induced downstream signaling. (A) 293T cells were cotransfected with the indicated cDNAs for 32 hours and serum starved for 16 hours. Phosphorylation of ERK and AKT, as well as total ERK, AKT, and β-actin, were detected by Western blotting. (B) REH cells were transduced with lentiviral particles encoding either scramble shRNA (nontargeting) or shRNA against Lnk (shLnk) and selected for 2 weeks with puromycin. Stable cells were serum starved overnight and stimulated with FL for 5 or 10 minutes, and then subjected to Western blotting using the indicated Abs. (C) 32D/FLT3-ITD cells were transfected for 32 hours with various Lnk constructs coexpressing GFP (a sorting marker) and serum starved for 16 hours. After fixation and permeabilization, cells were analyzed for GFP expression and phosphorylation of STAT5, AKT, or ERK using flow cytometry. The mean intensities of each phosphorylated molecule are shown. (D) 32D/FLT3-ITD cells were transfected with either MIG empty vector (MIG-EV) or MIG-Lnk for 48 hours and serum starved for 16 hours. GFP⁺ cells were purified using flow cytometry and subjected to real-time PCR analysis for Pim1 and Pim2 mRNA expression. Data represent the means ± SD of 2 independent experiments.