Expression and tyrosine phosphorylation of Kit and MAPK in ELM cells expressing KitD814H and wild-type Kit. (A) pcDNA3 vector (Invitrogen) and expression constructs for c-kit and c-kitD814H based on pcDNA3 were transfected into the Q2BN Quail fibroblastic cell line and expression of Kit protein was analyzed by Western blotting with anti-Kit antibodies. This blot was then stripped and an antiphosphotyrosine antibody was used. (B) Phosphorylation of the Kit receptor in ELM-I-1 cells and ELM-D cells stably transfected with wild-type c-kit or c-kitD814H expression vectors. Phosphorylation of the Kit receptor in unstimulated serum-starved cells was examined by immunoprecipitation with anti-Kit antibodies and antiphosphotyrosine Western blotting. The membrane was stripped and an anti-Kit antibody was used to verify Kit immunoprecipitation. (C) MAPK phosphorylation was analyzed in serum-starved ELM-I-1, ELM-Kit, or two clones of ELM-KitDH cells (i and ii) and in serum-starved ELM-Kit cells either stimulated for 10 minutes with 10 ng/mL rmSCF before lysis or continuously maintained in 10 ng/mL rmSCF. Whole cell lysates were analyzed by Western blotting using an antibody against the activated form of MAPK and a nonspecific antibody against p42/p44.