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. 2018 Jul 26;8:11287. doi: 10.1038/s41598-018-29371-0

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Reciprocal expression and functionality of human, chicken and chimeric TLR5 within chicken cellular background. Chicken HD-11 (A,B,C and E) or NF-κB reporter HD-11 cells (D) were nucleofected with expression plasmids coding for human (h), chicken (ch) or chimeric (ch/h) TLR5 from different species as indicated in the Methods. 24 h post nucleofection, selected wells were coincubated with purified recombinant Salmonella FliC (200 ng/well in 6-well plate format or 25 ng FliC in 96-well plate format). For quantitative RT-PCR, cells were stimulated for 30 min, and transcript amounts of chicken IL-8 (A,B) and IL-1β (C) were determined. Quantitated transcript values were normalized to respective chicken GAPDH transcript amounts and are presented in [%] relative to FliC-activated ch/hTLR5, set to 100%. In panel B, chIL-8 transcript induction in FliC-activated versus mock-coincubated non-transfected HD-11 cells is shown as fold induction. This result demonstrates a significant but considerably lower response of intrinsic chTLR5 in comparison to the activities provided by transfected TLR5 expression constructs shown in A and C. NF-κB-dependent luciferase activity was measured after 3.5 h of HD-11_luc stimulation using SteadyGlo luciferase assay (D). In D, the respective control values for all constructs (pEF6-V5 empty, hTLR5, chTLR5, ch/hTLR5) without FliC activation were subtracted from each FliC-activated value as background to yield the depicted activated end values for each construct in luminescence photon counts [C per s]. Mean and standard deviation from technical duplicates for transcriptional activation (A,C) and from technical triplicates for intrinsic TLR5 activity in HD-11 (B) and NF-κB-dependent activation (D) are shown. All experiments were independently repeated at least once, with similar outcomes. Significant differences between specific conditions are indicated by lines and asterisks in A, B, C and D (Student’s t-test; *0.01 < p < 0.05; ***p < 0.001; n.s non significant). Western Blot analysis for expression of TLR5 constructs using cleared lysates of nucleofected HD-11 cells (E). Immunoblotting was performed using anti-V5 or anti-actin antibody (loading control).