The D0 domain of flagellin mediates TLR5 activation through crosslinking.
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(A) A schematic representation of the domain organization of SaTy, SFΔD0, and a dimer of SFΔD0 tethered by peptide linkers. Numbering refers to S. typhimurium flagellin. (B) SDS-PAGE of purified recombinant proteins. (C) SFΔD0 is unable to activate TLR5 signaling, whereas activation with a tethered variant dimSFΔD0 has improved stimulation potential. (Above) Schematic representation of the role of the D0 domain in TLR5 activation. SFΔD0 (yellow) binds to but fails to activate TLR5 (cyan, magenta), whereas the linker in a dimer of SFΔD0 mimics the role of the D0 domain in crosslinking two ectodomains, therefore inducing active dimer formation. The human lung epithelial A549 cells were stimulated with SaTy flagellin, SFΔD0, or dimSFΔD0, and NF-κB-dependent SEAP activities were measured. (D) HEK293 cells transfected with a plasmid encoding hTLR5 were stimulated with increasing concentrations (0–1000 ng/ml) of recombinant proteins (SaTy, SFΔD0, or dimSFΔD0) and the NF-κB-dependent firefly and Renilla luciferase activities were measured 18 h later. (Data are representative of three (C) or two (D) independent experiments. Bars or points represent the means of 4 biological replicates ±s.d.; **p<0.005, nsp>0.05,) See also S4 Fig.