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. 2023 Mar 9;120(11):e2217602120. doi: 10.1073/pnas.2217602120

Fig. 2.

Fig. 2.

EmcB is sufficient to inhibit RLR signaling and prevents ubiquitination of RIG-I 2CARD. (A) Western blot analysis of GST-RIG-I 2CARD ubiquitination. GST-RIG-I 2CARD affinity purified from HEK293T cells coexpressing indicated protein for 48 h. Blot representative of four independent experiments. (B) Densitometric analysis of ubiquitination of GST-RIG-I 2CARD from A. P < 0.01 for − vs. NS1, − vs. EmcB, n.s. for − vs. EmcA by one-way ANOVA with Tukey’s post hoc test. (C) ISRE-luciferase signal from HEK293T cells transfected with GST-RIG-I 2CARD and indicated construct for 60 h. Luciferase signal normalized to no-GST-RIG-I 2CARD condition. Data are mean ± SD of three technical replicates representative of three independent experiments. P < 0.01 for − vs. NS1, − vs. EmcB; NS1 vs. EmcB n.s. by one-way ANOVA with Tukey’s post hoc test. (D) ISRE-luciferase signal from HEK293T cells transfected with increasing amounts of indicated pcDNA4 construct and poly(I:C). Luciferase signal normalized to no-poly(I:C) condition. Data are mean ± SD of four technical replicates representative of two independent experiments. Comparison n.s. for poly(I:C) vs. poly(I:C) + EmcB high by two-way ANOVA with Tukey’s post hoc test. (E) ISRE-luciferase signal from cells transfected with pcDNA4-FLAG-EmcB, poly(I:C) and MDA5 for 60 h. Data are mean ± SD of four technical replicates representative of two independent experiments. P < 0.0001 for poly(I:C), MDA5 vs. poly(I:C), MDA5, EmcB by two-way ANOVA with Tukey’s post hoc test.