Prolonged but not weak signals induced survival. (A) Kinetics of Erk phosphorylation after secondary cross-linking of CD8/γ. (i) FcRγ-deficient BMMCs expressing CD8/γ were cultured in the absence of IL-3 for 2 hours and stimulated with 3 μg/mL anti-CD8 or anti-CD8 plus goat antirat Ig for the indicated times. Total cell lysates were blotted with anti-phospho-Erk (upper panels) or anti-Erk (lower panels) to verify equal loading of proteins. (ii) The intensity of each band was quantified using LAS-1000 (Fuji, Tokyo, Japan) and expressed as arbitrary units. (B) Down-regulation of surface CD8/γ by secondary cross-linking. Cells were treated with anti-CD8 only or anti-CD8 plus goat antirat Ig Abs as described for panel A. At the indicated times, CD8/γ surface expression was assessed as described in “Materials and methods.” (C) Survival of CD8/γ-bearing BMMCs by secondary cross-linking. Cells were treated with anti-CD8 only or anti-CD8 plus anti–rat Ig Abs. Cell viability was determined similarly to Figure 1D. (D) Kinetics of Erk phosphorylation after stimulation with immobilized IgE(+Ag) in normal BMMCs. IL-3–starved FcRγ^+/+ BMMCs were stimulated either with 30 ng/mL DNP-HSA after one hour of sensitization (IgE(+Ag)), with plate-coated IgE(+Ag) as indicated in “Materials and methods” (immobilized IgE(+Ag)), or with 10 μg/mL soluble IgE (IgE(-Ag)). Erk phoshphorylation was determined similarly to panel A. (E-F) Degranulation and survival of normal BMMCs. BMMCs were stimulated with plate-coated Ag, plate-coated IgE(+Ag), or IgE similarly to panel D. After 4 days, histamine concentration in culture supernatant (E) and cell viability (F) were determined by ELISA and FACSCalibur, respectively. Data were means ± SDs of triplicate assays.