Fig 5. Active mutants of Flt3 (ITD and D835Y) transform human TF-1 cells to cytokine independent growth.

A) The human myeloid cell line TF-1 was transduced with recombinant retroviruses carrying wild-type (WT), ITD, or D835Y forms of Flt3, or with an empty vector as control. Following selection with G418, each of the TF-1 cell populations was cultured in the absence of GM-CSF, and viable cell outgrowth was monitored daily by CellTiter-Blue assay for 4 days. Each population was assayed in triplicate, and results are presented as the mean value ± the SD. B) Fes and Flt3 kinase proteins were immunoprecipitated from each of the cell populations shown in part A, followed by immunoblotting with antibodies to phosphotyrosine (pTyr), the Fes activation loop phosphotyrosine (pY713), as well as the Flt3 and Fes proteins. This experiment was performed in triplicate with comparable results, and a representative example is shown.