Figure 2.

Activation deficiency of chicken and porcine TLR5 can be partially rescued with respective TLR5 chimeras. HeLa (A) or HEK293-T cells (B) were transiently transfected with adjusted amounts of plasmid DNA encoding for TLR5 from different species or chimeric receptors (transfected plasmid amounts see Fig. 1). 48 h after transfection, cells were coincubated with purified recombinant Salmonella FliC (50 ng/well) or mock-treated for four hours. IL-8 secretion in the cell supernatants was determined by ELISA. IL-8 secretion of hTLR5-transfected, FliC-stimulated cells was set to 100% (reference); relative IL-8 secretion of all constructs with regard to the reference is depicted. Each condition was tested in two independent biological replicates (each in technical triplicates), the results of which are summarized here as mean and standard error. One representative experiment out of three is shown. For all constructs in A and B, except for pEF6-empty, the differences between mock-coincubated and FliC-coincubated condition were highly significant (Student’s t-test, unpaired, two-tailed; p < 0.01). Likewise, all differences between the full-length and respective chimeric constructs (in A) in the FliC-coincubated condition were also highly significant (p < 0.01). For clarity, asterisks here indicate solely the significant differences between FliC-activated hTLR5 and the corresponding activated full-length or chimeric constructs (Student’s t-test, unpaired, two-tailed) as follows: *0.01 < p < 0.05; **0.001 < p < 0.01; and ***p < 0.001, n.s. non-significant.