Figure 7.

Investigation of fatty acid binding to Toll-like receptor-4 (TLR-4)/MD-2 by using a FLAG-tagged extracellular part of TLR-4 fused to full-length MD-2 [lipopolysaccharide (LPS)-Trap]. The binding of ¹⁴C-labelled stearic and oleic acids to a soluble fusion complex consisting of the FLAG-tagged extracellular part of TLR-4 fused to full-length MD-2 via a flexible linker (LPS-Trap) was investigated by immunoprecipitation (upper panel) and by fatty acid binding studies (lower panel). The LPS-Trap was incubated with an excess of C¹⁴-labelled fatty acids or with LPS alone. The complex was immunoprecipitated with anti-FLAG antibody M2. LPS-Trap was incubated with biotin-labelled LPS with and without a 100 × molar excess of stearic or oleic acid, a dose that should compete effectively with LPS if specific fatty acid binding occurs. The LPS/LPS-Trap complex was then pulled down with streptavidin–agarose, Western-blotted, and the blot was probed with anti-FLAG antibody. Upper panel: Neither stearic acid nor oleic acid can bind to the TLR-4/MD-2 heterodimer. Lower panel: There was no specific binding of C¹⁴-labelled fatty acids to the TLR-4/MD-2 complex. IP= immunoprecipitation, WB= Western blotting.