Figure 5.

NF-κB reporter assays with WT and mutant MD-2. HEK293 cells were transfected with plasmid expression vectors for WT or mutant MD-2, TLR4, CD14, Firefly luciferase NF-κB reporter, and Renilla luciferase control. Twenty-four hours after transfection, cells were treated with 10 μM heme or 10 ng/ml LPS for 6 h in media containing 0.1% serum. After 6 h, cells were lysed and luciferase activity was measured and expressed as the ratio of Firefly luciferase NF-κB reporter to Renilla luciferase control. Luciferase activity is expressed as a percent of cells transfected with WT MD-2 stimulated with heme or LPS (100% activity, black bars and horizontal dashed black line). Magenta cluster and green cluster refer to the 2 clusters of potential heme binding amino acids in Figure 3. Results are representative of 3 independent experiments run in triplicate. Bar values are means ± SD. **P < 0.01 vs. WT MD-2 using Kruskal-Wallis One Way Analysis of Variance on Ranks.