Requirement of TSC1 IgE-induced mast cell degranulation. (A) Degranulation assessed by measuring release of β-hexosaminidase (β-hexo) from anti-DNP IgE-sensitized cells after stimulation with DNP-HSA at the indicated concentrations for 45 minutes. Data shown are mean ± SEM of triplicates and representative of 6 experiments. (B) Time course of mast cell degranulation using optimal Ag concentration (30 ng/mL). Data are representative of eleven experiments. (C-D) Impaired passive systemic anaphylaxis in TSC1 deficient mice. WT and TSC1KO mice were sensitized with anti-DNP IgE, after 24 hours, challenged with DNP-HSA, and the plasma histamine amounts (C) and mMCP-1 (D) were measured by ELISA (enzyme-linked immunosorbent assay) 90 seconds or 30 minutes after Ag challenge, respectively. (E) Impaired passive cutaneous anaphylaxis in TSC1 deficient mice. WT and TSC1KO mice were injected subcutaneously with IgE in ear, followed by DNP-HSA and Evan blue dye injection 24 hours later. The intensity of extracted dye from the ear collected 30 minutes after Ag challenge was measured by absorption at 610 nm (OD₆₁₀). Data shown are representative of 3 (C-D) and 2 (E) experiments (**P < .01; ***P < .001).