FcεRI-mediated signaling in TSC1-deficient mast cells. (A) Immunoblot analysis of whole-cell lysates of WT and TSC1KO BMMCs (exp indicates exposure). (B) Flow cytometry of the surface expression of FcεRI and c-Kit (solid lines). Dotted line indicates control Ig staining. (C) qRT-PCR analysis for FcεRI subunits, α, β, γ and chains. Bar graphs represent mean ± SEM (au indicates arbitrary unit). (D) mTORC1 signaling induced by FcεRI stimulation. BMMCs were sensitized for 4 hours with IgE in IL-3-free media and rested for 1 hour in Tyrode buffer before stimulation with DNA-HSA. Phosphorylation levels of mTORC1 signaling molecules were assessed by immunoblot analysis with specific anti–phospho-antibodies as indicated. (E) Akt, PKCδ, and MAPK signaling events after FcεRI-stimulation. Total protein was used as a loading control. Data are representative of 5 experiments.