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. 2023 May 9;220(8):e20220727. doi: 10.1084/jem.20220727

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© 2023 Lin et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 7. — ZNRF1 mediates TLR3 K63-linked polyubiquitination at K813 to inhibit type I IFN production and EMCV propagation. (A and B) HEK293T were cotransfected with Flag-TLR2 or Flag-TLR3 and ZNRF1-Myc. (C) HEK293T were cotransfected with HA-Ub, empty vector or TLR3-AcGFP or wild-type ZNRF1-Flag or ZNRF1 (C184A)-Flag for 36 h. Cell lysates were harvested and immunoprecipitated with the antibodies indicated. Immunocomplexes, as well as WCL, were subjected to immunoblotting (IB) with the antibodies indicated. (D) HEK293T cells were cotransfected with GFP-tagged ZNRF1, AcGFP-tagged TLR3, and HA-tagged various ubiquitin mutants; 36 h after co-transfection, a TLR3 ubiquitination assay was carried out by immunoprecipitating TLR3 and subsequent immunoblotting with anti-HA antibody. Quantification of TLR3 ubiquitination is shown in the lower panel of D. (E) HEK293T cells were cotransfected with the plasmids indicated for 36 h. Cell lysates were immunoprecipitated using anti-GFP antibodies. The immunoprecipitates were analyzed by immunoblotting using the antibodies indicated. (F) HEK293T cells were cotransfected with the plasmids indicated HA-Ub-K63, wild-type ZNRF1-Flag, or ZNRF1 (C184A)-Flag, and the indicated AcGFP-tagged wild-type TLR3 or TLR3 mutants. After 36 h, cell lysates were immunoprecipitated with the antibodies indicated. The immunocomplexes, as well as WCL, were subjected to immunoblotting with the antibodies indicated. (G and H) Tlr3^−/− iBMDM were reconstituted with either AcGFP-tagged wild-type TLR3 or TLR3(K813R) mutant. (G) The cell lysates were immunoprecipitated with anti-GFP antibodies. The immunocomplexes, as well as WCL, were subjected to immunoblotting with the antibodies indicated. (H) The expression of Ifnb mRNAs in iBMDMs after stimulation with poly(I:C) for the times indicated was analyzed by RT-qPCR. (I) The level of IFN-β in culture media after stimulation with poly(I:C) for the times indicated was measured by ELISA. (J) The level of IFN-β in culture media after infection with EMCV at MOI of 10 for the times indicated was measured by ELISA. (K) Cells were infected with EMCV at the MOI indicated for 24 h; viral titers in culture media were determined by plaque assay on Vero cells. (L) Quantification of EMCV virus particles in K. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test). Data are representative of three independent experiments (error bars, mean ± SD). Source data are available for this figure: SourceData F7.