TLR5 does not alter TLR4-dependent NF-κB activity in transfected HEK293 cells.

HEK293 cells stably expressing TLR4/MD-2/CD14 were transiently transfected with NF-κB-dependent firefly ELAM luciferase, and Renilla luciferase to permit detection of NF-κB activation by luciferase (ratio of ELAM/Renilla luciferase signal expressed as relative light units). A: Cells were stimulated with E. coli LPS for 4 hours. B: Cells were transfected with 5 ng TLR5 and 5 ng of pEF6 (empty vector), and stimulated with Salmonella Typhimurium flagellin at various concentrations for 4 hours. For both A and B, mean values ± standard deviations of triplicate conditions are displayed. Representative examples from four independent experiments are shown. C: Cells were transfected with increasing amounts of TLR5 and stimulated with 10 ng/mL E. coli LPS for 4 hours. Total DNA transfected was kept constant by the addition of pEF6 (empty vector). Data shown are the mean values ± standard deviations of three independent experiments normalized to relative light units for 0 ng TLR5 transfection.