Fig. 8.

Analysis of [³H]LOS transfer from [³H]LOS·albumin or [³H]LOS·sCD14 to His₆-sMD-2 or His₆-sMD-2·FLAG-TLR4_ecd. [³H]LOS capture to Ni²⁺ FF-sepharose was measured after incubation of increasing concentrations of [³H]LOS·albumin (A) or [³H]LOS·sCD14 (C) with either His₆-sMD-2 (0.2 nM) containing insect cell conditioned medium (total capture, --△--) or control insect cell conditioned medium lacking sMD-2 (non-specific capture, --▽--) in PBS (no Ca²⁺/Mg²⁺)/1% HSA for 3 h at 37°C. [³H]LOS co-capture to anti-FLAG agarose was measured after incubation of increasing concentration of [³H]LOS·albumin (B) or [³H]LOS·sCD14 (D) with medium containing His-₆-sMD-2·FLAG-TLR4_ecd (0.15 nM, total capture, --△--) or medium containing FLAG-TLR4_ecd (0.15 nM, non-specific capture, --▽--) alone. Specific capture (solid line) was calculated as the difference between total and non-specific capture and used for Scatchard analysis (figure inserts) as described in Experimental Procedures. Data shown are representative of > 3 independent experiments.