Constitutive tyrosine phosphorylation and activation of KIT^Del-Tyr814, but not of KIT^Del-Wild. (A) Flow cytometric analysis on the expression of KIT. To detect the expression of KIT on the cell surface, cells were incubated with either ACK2 MoAb (ECD) (—) or negative control antibody (---). To detect the whole expression of KIT, cells were fixed with methanol-aceton (1:1) and then incubated with either rabbit anti-KIT^Kinaseserum (Kinase) (—) or negative control antibody (---). (B) Constitutive tyrosine phosphorylation of KIT^Del-Tyr814. The state of tyrosine phosphorylation of KIT before and after stimulation with rmSCF was examined by immunoblotting using antiphosphotyrosine MoAb. The mobilities of KIT and deletion type of KIT are indicated at right. Three independent experiments were performed with comparable results. (C) Constitutive activation of KIT^Del-Tyr814. KIT was immunoprecipitated from cell lysates without rmSCF stimulation using anti-KIT^Kinase serum. The immunoprecipitated KIT was examined by immunoblotting using rabbit Ab-1 (upper panel) and was subjected to immune complex kinase assay (lower panel). Three independent experiments were performed with comparable results.