FIGURE 9.

CD14 binds to solid phase lipids. A, affinity-purified preparations (2 μg each) of the extracellular domains of CD14 (sCD14) and TLR4 (sTLR4) and the full-length MD-2 were analyzed by gel electrophoresis under reducing and denaturing conditions. The electrophoretic gels were stained with Coomassie Blue. mol. mass st., molecular mass standards. B, phospholipids (1.25 nmol) in 20 μl of ethanol were placed onto microtiter wells, and the solvent was evaporated. Nonspecific binding was blocked with 20 mm Tris buffer (pH 7.4) containing 0.15 m NaCl, 5 mm CaCl₂ (in the upper panel), or 2 mm EGTA (in the lower panel) and 5% (w/v) bovine serum albumin (buffer A). Varying concentrations of human CD14 in buffer A were added and incubated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using anti-CD14 monoclonal antibody as described under “Experimental Procedures.” The data shown are the means ± S.E. from three separate experiments with duplicate samples in each experiment.