Different activation of STAT5 by FLT3-ITD and FLT3-TKD mutants. (A) Ba/F3 and (B) 32D cells were stably transfected with WT or mutated FLT3 constructs as indicated. All cells were serum starved for 4 hours. Parental cells (par) and FLT3-WT-expressing cells were either untreated or stimulated with IL-3 or FLT3 ligand (FL), respectively. FLT3 was immunoprecipitated (IP) from whole-cell lysates and immunoblotted with antiphosphotyrosine antibody and reblotted with anti-FLT3 antibody. Activation of downstream targets is demonstrated by immunoblotting the total cell lysates with the indicated phosphospecific antibodies. After stripping the membrane was reprobed with indicated total antibodies to demonstrate equal loading. (C) Bone marrow of 5-FU-pretreated mice was transduced with equivalent retroviral titers of the indicated constructs. After the infection procedure the cells were washed twice with phosphate-buffered saline (PBS) and subsequently starved for 4 hours in medium containing 2% FCS and no cytokines. After FACS analysis of the cells (left), total cell lysates were prepared and separated by SDS-PAGE. Expression of FLT3 protein and activation of downstream targets is demonstrated by immunoblotting the total cell lysates with the indicated phosphospecific antibodies. After stripping the membrane was reprobed with indicated total antibodies to demonstrate equal loading. In left panels, circles indicate EGFP⁺ cells; numbers, the percentage of EGFP⁺ cells.