IgE-mediated cytokine production in TSC1-deficient mice. (A) In vivo cytokine production on IgE-mediated PSA. Mice were sensitized and challenged as described in Figure 2C. IL-6 and TNFα in plasma 180 minutes after challenge were determined by ELISA. (B) ELISA for IL-6 and TNF-α production by BMMCs. IgE-sensitized cells were cross-linked with 30 ng/mL DNP-HSA (Ag) for 6 and 24 hours. (C) qRT-PCR analysis of mRNA encoding IL-6 and TNF-α after 1 and 3 hours of stimulation with Ag. (D) Effects of rapamycin on cytokine production. WT and TSC1KO BMMCs were stimulated with DNP-HSA for 6 hours in the presence or absence of rapamycin as indicated, and IL-6 and TNF-α in supernatants were measured by ELISA. (E) qRT-PCR of IL-6 and TNF-α transcripts after 1 hour of stimulation with DNP-HSA in the presence or absence of rapamycin. Data shown are representative of 5 experiments and bar graphs display mean ± SEM (au indicates arbitrary unit; *P < .05; **P < .01; ***P < .001).