American Society of Hematology

Figure 3.

$Figure 3. PKCδ associated with membranes from IC2 cells expressing D814Y Kit has significantly greater kinase activity. In vitro immune complex kinase assays were performed using membrane-enhanced fractions from cells expressing wild-type or D814Y Kit. (A) IC2 cells expressing WT or D814Y Kit were stimulated with 100 ng/mL murine SCF for 15 minutes at room temperature prior to isolation of membranes. Membrane protein (400 μg per sample) was immunoprecipitated with an antibody specific for PKCδ. The immunoprecipitates were incubated with γ32PATP, phosphatidylserine, and histone H1 as a substrate. Samples were incubated for 20 minutes at room temperature before resolution by SDS-PAGE and transfer to immobilon-P. Substrate phosphorylation was visualized by exposure to autoradiography film. (B) Membranes were immunoblotted with an antibody specific for PKCδ. These data are representative of results from more than 3 experiments.$

PKCδ associated with membranes from IC2 cells expressing D814Y Kit has significantly greater kinase activity. In vitro immune complex kinase assays were performed using membrane-enhanced fractions from cells expressing wild-type or D814Y Kit. (A) IC2 cells expressing WT or D814Y Kit were stimulated with 100 ng/mL murine SCF for 15 minutes at room temperature prior to isolation of membranes. Membrane protein (400 μg per sample) was immunoprecipitated with an antibody specific for PKCδ. The immunoprecipitates were incubated with γ^32PATP, phosphatidylserine, and histone H1 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.

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