The 787F mutation impairs c-Kit downstream signaling. (A) BMMCs from wild-type (wt) and Kit^787F (787F) mice were stimulated for the indicated times with SCF (100 ng/mL). Cell lysates were immunoblotted with antibodies specific for (phospho-)Erk1/2 and p38 (A) or (phospho-)c-Kit, PLCγ1, p90Rsk, Cbl, and actin (B). (C) IgE-preloaded BMMCs from wild-type (wt) and Kit^787F (787F) mice were stimulated for the indicated times with antigen (DNP-HSA; 50 ng/mL). Lysates were immunoblotted with antibodies specific for (phospho-)Erk1/2. (D) BMMCs from wild-type and Kit^787F mice were stimulated with SCF (100 ng/mL), and calcium mobilization was measured. The arrow marks the SCF addition. BMMCs generated from 3-5 mice of each genotype were analyzed. Comparable results were obtained with BMMCs from different cultures. Representative curves are shown. At least 3 independent experiments were performed. (E) BMMCs from wild-type (wt) and Kit^787F (787F) mice were stimulated for the indicated times with SCF (100 ng/mL). Cellular lysates were subjected to immunoprecipitation using an antibody against c-Kit, and immunoprecipitated c-Kit was analyzed by immunoblotting for ubiquitin. Immunoblotting with c-Kit–specific antibodies served as a loading control.