Expression of KITD814V results in constitutive phosphorylation of KIT and ligand-independent growth. (A) Schematic diagram of retroviral vectors expressing WT KIT and KITD814V. (B) KITD814V is constitutively autophosphorylated in the absence of growth factor stimulation. 32D cells expressing vector alone, WT KIT, or KITD814V were lysed and equal amounts of protein were subjected to immunoprecipitation using an anti-KIT antibody followed by Western blot analysis using an antiphosphotyrosine antibody. Shown is the level of phosphorylation and total KIT protein in each lane. (C) Expression of KITD814V in 32D cells induces ligand-independent growth. 32D cells expressing empty vector, WT KIT, or KITD814V were subjected to a thymidine incorporation assay in the absence of growth factor stimulation. Bars denote the mean thymidine incorporation (cpm ± SD) from 1 of at least 3 independent experiments performed in quadruplicate. *P < .05, KITD814V versus vector or WT KIT.