FIG. 7.

Examination of the interaction of 3ECD with poly(I-C) and ODN2006. (A) Recombinant 3ECD binding to ODN2006 and poly(I-C) in a UV-induced cross-linking (X-link) assay. ODN2006 and IC18 were radiolabeled by a kinase reaction, and the autoradiograph is shown in the upper panel. The lower panel contains a Coomassie (Cooma.) blue-stained gel that is intended to allow normalization for the amount of 3ECD used in each reaction. The competitors (Comp.) used were mock (φ), ODN2006, and heterogeneous poly(I-C). “M” corresponds to the size marker. (B) ODN2006 is a potent inhibitor of TLR3 signaling even at higher poly(I-C) concentrations. Since the experiment was performed with different concentrations of ligands, the basal level of luciferase activity is expected to change. Hence, the ratios of the levels of NF-κB-driven firefly luciferase to the levels of the putatively constitutive thymidine kinase promoter are graphed. The level of induction is listed below the graph. Note that there is a modest increase in TLR3 signaling at 10 and 20 μg/ml of poly(I-C) in comparison to the level at 2.5 μg/ml of poly(I-C). The means of the results are shown above the bars, and the error bars show standard errors of the means. pIC, poly(I-C); 2006, ODN2006; +, present; −, absent.