Figure 5. Binding to human MD-2 of C. canimorsus lipid A depends on the core-oligosaccharide.

Soluble human MD-2 from cell culture supernatant was combined with the indicated mixture of E. coli LPS-Biotin and a competitor (either C. canimorsus lipid A, lipid A-core, E. coli O111 LPS, penta-acyl E. coli lipid A or lipid IV_A). Biotinylated E. coli LPS-MD-2 complexes were purified and analyzed by non-reducing, denaturing Western blotting for presence of MD-2. (A) Untreated human MD-2 did not bind to the Strep-column (lane 1), addition of E. coli LPS-biotin lead to co-purification of human MD-2 (lane 2). Results shown are representative of three independent determinations. (B) Quantification of Western-blots as shown in A. Values are shown as percentage of the corresponding positive control. Data were combined from n = 3 independent experiments, error bars indicated are standard error of the mean. (C) as in (A) but the C. canimorsus lipid A stock solution was pretreated with either 0.1% TEN or 50% DMSO and sonication in both cases to increase its solubility in water/buffer.