Ligand-independent activity of KIT mutants in Ba/F3 cells. (A) Tyrosine phosphorylation of KIT mutants in Ba/F3 cells. Whole-cell lysates obtained from cells unstimulated or stimulated for 5 minutes with 250 ng/mL mSCF were subjected to SDS-PAGE and immunoblotted with anti-PY⁷¹⁹KIT antibody. The blot was stripped and reprobed with anti-KIT antibody. Arrows indicate the position of mature (top arrow) and immature (bottom arrow) forms. (B) Cytokine-independent proliferation of Ba/F3 cells carrying KIT mutants. Cells were exposed to 250 ng/mL mSCF or 12.5 ng/mL IL-3 or left unstimulated for 48 hours. Cell proliferation was assessed by measuring mitochondrial conversion of WST-1 into blue formazan dye with a spectrophotometer. Data are shown as percentage of absorbance at 450 nm compared with absorbance of the respective cell line under IL-3 stimulation. (C) Effect of tyrosine kinase inhibitors on ligand-independent proliferation of Ba/F3 cells. Ba/F3 cells expressing WT were introduced in a proliferation assay and incubated for 48 hours with 250 ng/mL mSCF or 12.5 ng/mL mIL-3 and without growth factor for KIT mutants expressing cells and in the presence of 1μM imatinib or dasatinib. Values are presented relative to cell proliferation in the absence of inhibitors. (B-C) Data are mean ± SD of triplicates. Results shown are representative of 3 independent experiments.