FIGURE 4.

Ionic interactions between lipid IV_A and TLR4 at the dimerization interface are essential for lipid IV_A responsiveness. HEK293 cells were transiently transfected with: 1) one of the four constructs: mTLR4^YFP wild type construct (WT), the mTLR4^YFP K367E/R434Q mutant (K367E/R434Q), hTLR4^YFP wild type construct, and the hTLR4^YFP E369K/Q436R mutant (E369K/Q436R); 2) NF-κB luciferase plasmid; and 3) a Renilla luciferase plasmid for luciferase assay. After overnight transfection, the cells were stimulated with indicated concentrations of LPS·lipid IV_A and mMD-2 (200 ng/ml) under serum-free conditions. Luciferase activity was measured in cell lysates the next day. The data are reported as the means ± S.D. of three independent wells for each data point. The luciferase activities were normalized from Renilla luciferase activity. One representative data set from four replicates is shown in the figure.