Figure 1

NF-kB dependent signalling from human and bovine TLR5 clones in HEK293 cells. HEK293 cells were transfected with an NF-κB-dependent secreted alkaline phosphatase (SEAP) reporter plasmid alone (Null cells) or with the reporter in combination with either a human TLR5 (hTLR5) or bovine (bTLR5) expression plasmid. SEAP levels were determined following addition of different concentrations of WT-H7 flagellin or site-directed flagellin mutants. (A) NF-kB dependent SEAP production from bTLR5 and hTLR5 following overnight stimulation with a range of H7 flagellin concentrations. SEAP activity from cells transfected with either TLR5 construct was significantly higher than the non-transfected control at levels of 0.5 ng/mL flagellin or greater (p < 0.005). hTLR5 produced higher SEAP activity compared to bTLR5 at two concentrations of flagellin, 0.05 and 0.5 ng/mL (p = 0.027 and p < 0.001 respectively). (B) NF-kB dependent SEAP production from bTLR5 in HEK293 cells stimulated overnight with different concentrations of WT-H7 and the indicated flagellin mutants. The dashed line indicates the response to WT-H7. (C) NF-kB dependent SEAP production from hTLR5 in HEK293 cells following addition of WT H7 and an R90T variant. Differences of p < 0.001 are indicated by an asterisk. All experiments in this figure were performed a minimum of three times with at least three technical repeats. The data shown are the means and 95% confidence intervals.