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. Author manuscript; available in PMC: 2012 Jul 15.
Published in final edited form as: J Immunol. 2011 Jun 17;187(2):692–701. doi: 10.4049/jimmunol.1003658

FIGURE 1.

FIGURE 1

Gamma secretase inhibitors (GSI) significantly down-regulate Th17 associated cytokine levels in murine Th17 in-vitro polarization assays. (A) ELISA for IL-17A, IL-17F and IL-21 in supernatants of activated CD4+ T cells from C57BL/6 mice. Cells were pretreated in-vitro with GSI (25μM ILCHO and 4μM Compound E) or with 0.1% DMSO (as a vehicle control) before 24, 48 and 72 hours culture in Th17 polarizing conditions. Cells were then lifted, recounted and cultured overnight. (B) Notch1 expression in cells pretreated with or without ILCHO was evaluated by immunoblotting using antibodies that recognized the cleaved active Notch1. Antibody specific for actin was used to control for loading. (C) Evaluation of Notch1 expression in cells pretreated with or without ILCHO by flow cytometry using antibodies specific for CD4+ cells and intracellular Notch 1 (Notch1IC). (D) Intracellular staining of IL-17 and IFNγ in Th17 differentiated cells treated with either DMSO or GSI. (E) Naïve CD4+ T cells were differentiated towads Th17 subset for 4 days followed by treatment with either DMSO or GSI. Supernatants were collected after 24 h and IL-17 A ELISA was performed. Data shown here represents one of at least three independent experiments done in triplicates.*p≤0.05, **p≤0.001, ***p≤0.0001.