PKCδ is constitutively active in cells expressing D814Y Kit. In vitro immune complex kinase assays were performed using whole cell lysates from cells expressing wild-type (WT) or D814Y Kit. (A) IC2 cells expressing wild-type or D814Y Kit were stimulated with 100 ng/mL murine SCF for 15 minutes at room temperature prior to lysis. Whole cell lysate protein (800 μg per sample) was immunoprecipitated with an antibody specific for PKCδ. The immunoprecipitates (IP) were incubated with γ^32PATP, phosphatidylserine, and myelin basic protein (MBP) as a substrate. Samples were incubated for 20 minutes at room temperature before resolution by SDS-PAGE and transfer to immobilon-P. Substrate phosphorylation was visualized by exposure to autoradiography film. (B) Membranes were immunoblotted with an antibody specific for PKCδ. These data are representative of results from more than 3 experiments.