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. 2018 Oct 25;9:2479. doi: 10.3389/fimmu.2018.02479

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Copyright © 2018 Liu, Li, Song and Xu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Overexpression of TRIM21 activates the virus-induced type I IFN signaling via catalyzing the K27-linked polyubiquitination of MAVS and increases IRF3 phosphorylation. (A) HeLa cells were transfected with TRIM21 expression or vector plasmids. 24 h after transfection, cells were infected with CVB3 (MOI = 5) for the indicated time. The mRNA levels of endogenous IFN-β and IFN-α were detected by Q-PCR assay. Data were presented as mean ± SEM of three representative independent experiments. (B) HEK293 cells were co-transfected with the IFN-β promoter reporter plasmids and the indicated amounts of TRIM21 expression plasmid or 100 ng vector plasmid. 24 h later, the luciferase activity was determined. HEK293 cells were co-transfected with the IFN-β promoter reporter plasmids and TRIM21 expression plasmid or mock vector. 24 h later, cells were infected with CVB3 (MOI = 5) for 12 h and the luciferase activity was determined. HEK293 cells were co-transfected with the IFN-β promoter reporter plasmids and TRIM21 expression plasmid or mock vector, together with MAVS or MDA5 expression plasmids. 24 h later, cells were infected with CVB3 (MOI = 5) for 12 h and the luciferase activity was determined. Data were presented as mean ± SEM of three representative independent experiments. (C) HeLa cells were transfected with TRIM21 expression or vector plasmids for 24 h before infection with CVB3 (MOI = 5). The mRNA levels of endogenous ISG15 and ISG54 were detected by Q-PCR assay. (D) HEK293 cells were co-transfected with the IRF3 promoter reporter plasmids and TRIM21 expression plasmid or mock vector. 24 h later, cells were infected with CVB3 (MOI = 5) for 12 h and the luciferase activity was determined and data were presented as mean ± SEM of three representative independent experiments. (E,F) HeLa cells were transfected with TRIM21 expression or vector plasmids for 24 h before infection with CVB3 (MOI = 5). Cell lysates were subjected to probe withanti-IRF3, anti-pIRF3 and anti-GAPDH antibodies by Western blotting and Native page for the detection of IRF3 dimerization. (G) HeLa cells were transfected with Flag-MAVS plasmids as indicated, 24 h later cells were infected with CVB3 (MOI = 5). Cellular lysates were immunoprecipitated with anti-Flag. Immunoprecipitates were analyzed by WB with anti-Flag and anti-TRIM21. (H) HeLa cells were co-transfected with TRIM21, Flag-MAVS, and HA-K27Ub for 24 h and treated with CVB3 for additional 12 h. Ubiquitination and immunoblotting assays were performed with indicated antibodies. *p < 0.05; **p < 0.01; ***p < 0.001.