FIGURE 2.

LBP and soluble CD14 enhance soluble TLR1·TLR2·Pam₃CSK₄ ternary complex formation but are not part of the final ternary complex. In A–D, various combinations of 0.25 μm TLR1, 0.05 μm TLR2, 2.5 μm Pam₃CSK₄, 0.05 μm LBP, and/or 0.25 μm sCD14 were incubated for 2 h at 37 °C in a 500-μl volume of PBS buffer, pH 7.4 as indicated. Protein complexes were separated by size exclusion chromatography. The expected molecular weight of the TLR monomers and dimers was estimated by column calibration using known molecular weight standards. Proteins in eluted fractions were separated by 7.5% SDS-PAGE and transferred by Western blotting, and TLR1, TLR2, LBP, and sCD14 were detected using suitable antibodies and HRP conjugates (see “Experimental Procedures”). The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.