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. 2012 Jul 24;287(37):30993–31002. doi: 10.1074/jbc.M112.376186

FIGURE 5.

FIGURE 5.

Fusion of a designed coiled-coil with mTIR creates inhibitor with broad TLR and IL-1R inhibitory spectrum. A–C, GCN-mTIR is a strong inhibitor of TLR5, TLR9 and TLR3 signaling. HEK293 cells were transfected with mTIR, cc-mTIR or GCN-mTIR plasmids (1, 5, 10 ng) and either TLR5 plasmid (20 ng) (A), TLR9 plasmid (10 ng) and Unc93B1 plasmid (1 ng) (B) or TLR3 plasmid (20 ng) (C). Cells were stimulated with flagellin (10 ng/ml), ODN (3 μm) or poly(I:C) (10 μg/ml). D, GCN-mTIR inhibits signaling triggered by overexpression of TRIF. Cells were transfected with control or TRIF plasmid (2 ng) or cotransfected with TRIF (2 ng) and GCN-mTIR plasmid (10, 30, 50 ng). E, coiled-coils linked TIR proteins improve inhibition of IL-1R signaling. HEK293 cells were transfected with mTIR, cc-mTIR or GCN-mTIR plasmids (1, 5, 10 ng) and stimulated with IL-1β (50 ng/ml). F, TNFα-R signaling is unaffected by TIR fusion proteins. HEK293 cells were transfected with mTIR, cc-mTIR or GCN-mTIR plasmids (5, 35, 75 ng) and stimulated with TNF-α (100 ng/ml). Activation of TLR signaling pathway was determined by the dual luciferase assay. Representative graphs are shown from three separate experiments. Data on graphs are represented as mean ± S.D.