Mutation of galU does not cause gross changes in O-antigen synthesis, serum sensitivity, or TLR signaling. Panel A: Bacterial cell lysates (10 μg/lane) and LPS preparations of WT, galU mutant, and wbtA-mutant (O-antigen deficient) FT strains were subjected to SDS-PAGE and Western blotting using an FT LPS-specific monoclonal antibody preparation. Panel B: HeLa-TLR4/MD-2 or HeLa-TLR2 were transiently transfected with a ELAM-luciferase reporter construct, CMV-CD14 and CMV-β-Gal (for normalization) and stimulated for 6 hours with 2μg or 10μg of the indicated FT lysates. NF-κB activation was measured via a luciferase assay. Statistical analyses were performed via one-way ANOVA and significant differences (P < 0.0001) are indicated (***). Panel C: Bacteria were incubated for 2 h at 37°C in either PBS alone or PBS containing either 10% or 20% fresh frozen human plasma (FFP) or 20% heat-inactivated (HI-FFP). Viable bacteria were then enumerated by dilution plating. 100% viability was defined as the number of bacteria recovered from PBS containing no serum (0% FFP), and results were plotted as the mean (± SEM) of triplicate samples. Statistical analyses were performed via one-way ANOVA and statistically significant differences (P < 0.0001) are indicated (***).