FN binding enhances IgE-induced intracellular signaling events, cytokine production, and survival. (A) Total cell lysates (50 μg as assessed by BCA assays) from IgE-stimulated suspension and FN-adhered BMMCs were subjected to Western analysis using anti-phosphotyrosine antibodies (4G10) (top panel) or antiphospho-specific Erk antibodies (middle panel) and reprobed with anti-Erk antibodies (bottom panel) to demonstrate equal loading. (B) BMMCs were stimulated for 3 hours at the indicated concentrations of IgE in the presence of 15 μ polystyrene beads coated with BSA (□) or FN (▪). IL-6 (left panel) and TNF-α (right panel) levels in the supernatants were detected by ELISA. (C) BMMCs were plated at 5 × 105 cells/mL in IMDM + 0.1% BSA ± IgE at 2 and 5 μg/mL in FN (▪) or BSA (□) coated wells. On day 4, viable cells were counted by trypan blue exclusion (left panel). In the right panel, BMMCs were set up as above ± 2 μg/mL IgE and viable cells counted on day 7. Data points are the mean ± SEM of 6 (B), 2 (C, left) and 3 (C, right) determinations. Similar results were obtained in 3 (A), 4 (B), and 3 (C) separate experiments.
