Expression of D816V c-Kit in MIHC results in constitutive tyrosine phosphorylation of multiple proteins.

(A) Human c-Kit protein is expressed in MIHCs infected with wild-type or D816V c-Kit. Equivalent amounts of protein from clarified lysates of parental MIHCs or MIHCs infected with the indicated human c-Kit construct were immunoprecipitated with antibody specific for c-Kit, resolved with SDS-PAGE, transferred to Immobilon, and immunoblotted with antiserum specific for c-Kit. (B) Comparison of phosphotyrosyl proteins in MIHCs expressing wild-type or D816V c-Kit. Cells were incubated for 10 minutes with or without huSCF (100 ng/mL) as indicated, lysed, and immunoprecipitated (IP) with antibody specific for phosphotyrosine (PY-20). Samples were resolved using SDS-PAGE, transferred to Immobilon, and immunoblotted (IB) with antibody specific for phosphotyrosine (4G10). Phosphotyrosyl proteins were visualized with enhanced chemiluminescence (ECL).