Fig. 1.

Induction of NF-κB by zebrafish TLR5b (drTLR5b) and drTLR5a upon stimulation with purified flagellins and bacterial lysates. (A and C–H) HeLa-57A cells were transfected with drTLR5b, drTLR5a, hTLR5 or drTLR5b and drTLR5a combined as indicated. Control cells were transfected with empty vector. Cells were stimulated (5 h) with vehicle (–) or 1 µg mL⁻¹ of purified recombinant FliC^SE or FliC^PA flagellin (A, C, E, and G) or 2 µg mL⁻¹ of total protein from lysates of S. Enteritidis, P. aeruginosa,or A. hydrophila (D, F, and H). Note that the NF-κB response of the drTLR5 heterodimer is higher when stimulated with bacterial lysate compared with purified recombinant flagellin. (B) Fluorescence microscopy of empty vector or FLAG-tagged drTLR5b transfected HeLa-57A cells stained with M2 α-FLAG and DAPI for nuclear visualization. (White scale bars: 10 µm.) NF-κB activity is represented by luciferase activity in RLUs. Values are the mean ± SEM of three independent experiments (A, C, E, and G) or a representative of three independent experiments (D, F, and H) all performed in duplicate.