The 85-kd regulatory subunit of PI3K is constitutively associated with D816V c-Kit.
(A) p85PI3K coimmunoprecipitates with D816V c-Kit. Indicated cells were incubated in the presence or absence of huSCF for 10 minutes and lysed. Equivalent amounts of protein from the clarified lysates were immunoprecipitated with antisera specific for c-Kit or a matched control antibody, idicated by C. Immunoprecipitates were washed, proteins were resolved with SDS-PAGE and transferred to Immobilon, and the lower portion of the membrane was immunoblotted (IB) with antisera specific for p85PI3K. Proteins were visualized with ECL. (B) Comparison of the expression levels of wild-type and D816V c-Kit in MIHCs. The upper portion of the membrane from panel A was immunoblotted with antisera specific for human c-Kit. (C) The 85-kd regulatory subunit of PI3K is constitutively phosphorylated in cells expressing D816V c-Kit. Indicated cells were incubated in the presence or absence of huSCF for 10 minutes and lysed. Equivalent amounts of protein from the clarified lysates were immunoprecipitated with antisera specific for p85PI3K or a matched control antibody, indicated by C. Immunoprecipitates were washed, and proteins were resolved with SDS-PAGE and transferred to Immobilon. The membrane was immunoblotted (IB) first with antisera specific for phosphotyrosine (upper panel), then stripped and reprobed with antibody specific for p85PI3K (lower panel).
