FIGURE 3.

Dasatinib inhibits the constitutive ubiquitination of c-Kit/D816V and increases the cell surface expression of the receptor. Ba/F3 cells expressing wild-type (wt) c-Kit or c-Kit/D816V mutant were incubated with or without Dasatinib (2 nm) for 24 h. A, cells were starved and stimulated with SCF (100 ng/ml) and subjected to immunoprecipitation using a c-Kit antibody. Levels of ubiquitination, tyrosine phosphorylation, and c-Kit expression were analyzed by Western blot (IB). pY, phosphotyrosine. B, cells were stained with PE-conjugated anti-c-Kit antibody or isotype control followed by examination of expression using flow cytometry. Cell surface expression levels of c-Kit were quantified, and the expression level of wild-type c-Kit without incubation with Dasatinib was considered as 100% and compared with other cells (gray column: no incubation; black column: incubation with Dasatinib). The data shown are the average of three independent experiments with error bars defining the standard deviation.