Fig. 2.

Oligomerization state and ligand binding of TLR8^Z-loop. (A) Gel filtration chromatography of TLR8^wt and TLR8^Z-loop. TLR8^wt (Left, Top), TLR8^wt/ORN06 (Left, Middle), TLR8^wt/R848 (Left, Bottom), TLR8^Z-loop (Right, Top), TLR8^Z-loop/ORN06 (Right, Middle), and TLR8^Z-loop/R848 (Right, Bottom) were analyzed by Superdex 200 gel filtration chromatography. The black and gray lines show absorbance at 280 nm (A₂₈₀) and 260 nm (A₂₆₀), respectively. Molecular weights estimated from elution volume are shown above the peak. The ratios of A₂₆₀ to A₂₈₀ are given in parentheses. (B) ITC analysis of TLR8^Z-loop titrated with R848 (Left) and ORN06 (Right). (C) NF-κB activation by TLR8^WT and TLR8^Z-loop. The response to agonistic ligand under synergistic conditions was assessed using NF-κB–dependent luciferase assay. To induce synergy, cells were treated for 6 h by indicated TLR8 ligands. Data represent the mean fold induction of NF-κB activity, calculated as the RLU of stimulated cells divided by the RLU of nonstimulated cells. Data from three independent experiments are shown.