Self-association of KIT^Tyr814. (A) 293T cells were transfected with pEF-BOS containing c-kit^Wild, pEF-BOS containing c-kit^W42, pEF-BOS containing c-kit^Del-Wild, or pEF-BOS containing c-kit^Del-Tyr814 and cotransfected with pEF-BOS containing c-kit^Del-Wild and pEF-BOS containing c-kit^Wild, pEF-BOS containing c-kit^Del-Wild and pEF-BOS containing c-kit^W42, pEF-BOS containing c-kit^Del-Tyr814 and pEF-BOS containing c-kit^Wild, or pEF-BOS containing c-kit^Del-Tyr814 and pEF-BOS containing c-kit^W42. The expression of KIT is shown in upper panel. Cell lysates were immunoprecipitated with rabbit anti-KIT^Kinase serum (Kinase), and then the immunoprecipitates were subjected to SDS-PAGE for 90 minutes and immunoblotting with rabbit Ab-1. The phosphorylation bands of immune complex kinase assay are shown in lower panel. KIT was immunoprecipitated from cell lysates with ACK2 MoAb (ECD), and the immunoprecipitates were incubated with γ-[³²P]-ATP. Incorporation of ³²P into KIT and/or Del-KIT was visualized by autoradiography. (B) 293T cells were transfected with pEF-BOS containing c-fms/c-kit^Wild or pEF-BOS containing c-fms/c-kit^Tyr814 and cotransfected with pEF-BOS containing c-fms/c-kit^Wild and pEF-BOS containing c-kit^W42 or with pEF-BOS containing c-fms/c-kit^Tyr814 and pEF-BOS containing c-kit^W42. The expression of FMS/KIT and/or KIT^W42 is shown in upper panel. Cell lysates were immunoprecipitated with rabbit anti-KIT^Kinase serum (Kinase) before and after treatment with rhM-CSF, and then the immunoprecipitates were subjected to SDS-PAGE for 3 hours and immunoblotting with rabbit Ab-1. The phosphorylation bands of immune complex kinase assay are shown in lower panel. KIT was immunoprecipitated from cell lysates with 3-4A4 MoAb (FMS-ECD), and the immunoprecipitates were incubated with γ-[³²P]-ATP. Incorporation of ³²P into FMS/KIT and/or KIT^W42 was visualized by autoradiography. The similar results were obtained from three independent experiments.