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Fig. 4 Figure 4. OTUD3-deficiency enhances RLR signaling in MLFs (A and B) qRT-PCR analysis of Ifnb1, Isg56, and Cxcl10 in Otud3+/+ and Otud3−/− MLFs un-infected or infected with SeV or EMCV for the indicated times. (C) qRT-PCR analysis of Ifnb1, Isg56, and Cxcl10 in Otud3+/+ and Otud3−/− MLFs transfected with poly(I:C) for the indicated times. (D) qRT-PCR analysis of Ifnb1, Isg56, and Cxcl10 in Otud3+/+ and Otud3−/− MLFs transfected with viral or synthetic RNAs for the indicated times. (E and F) ELISA of IFN-β in supernatant of Otud3+/+ and Otud3−/− MLFs infected with VSV or EMCV for 6–12 h. (G–I) IB of proteins in Otud3+/+ and Otud3−/− MLFs un-infected or infected with SeV, VSV, or EMCV for the indicated times by the indicated antibodies. (J) Fluorescence microscopy imaging of the replication of VSV-GFP in Otud3+/+ and Otud3−/− MLFs followed by the infection of VSV-GFP for 16 h. Scale bar, 200 μm. (K) IB of viruses-infection efficiency in Otud3+/+ and Otud3−/− MLFs followed by the infection of VSV-GFP for 16 h. (L) qRT-PCR analysis of VSV mRNA in Otud3+/+ and Otud3−/− MLFs followed by the infection of VSV-GFP for 16 h. (M) Plaque assay for VSV-GFP titers in Otud3+/+ and Otud3−/− MLFs followed by the infection of VSV-GFP for 16 h. (N) IB of reconstituted gene expression. (O–R) qRT-PCR analysis of Ifnb1 and Il6 in Otud3−/− BMDCs reconstituted with the empty phage (control), OTUD3, or OTUD3-C76A followed by infected with SeV or EMCV for 0–6 h. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, using unpaired Student’s t test (L and M) or two-way ANOVA with Holm-Sidak’s multiple comparisons test (A–F and O–R). Data based on one representative experiment performed in three biological replicates from at least three independent experiments (mean ± SD) or representative data (G–I, J, K, and N).