plasmid ubr5 fl  (Addgene inc)

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a Immunoblots of UBR5 protein in wild type (WT) and three UBR5 −/− (made with unique guide RNA) in 2fTGH-ISRE-Luc cells. Quantification of the ( b ) intracellular ISRE-Luc activity, c secreted IFN-β protein by ELISA, d cellular IFNB1 mRNA levels in 2fTGH-ISRE-Luc cells transfected with poly (I:C) for 12 h. Quantification of the ( e ) intracellular ISRE-Luc activity, f secreted IFN-β protein, g cellular IFNB1 mRNA, h secreted type I IFNs (Bioassay) in WT, UBR5 −/− (made with gRNA #1) and IFIH1 −/− (gene symbol for MDA5) HEK293T cells transfected with poly (I:C) for 12 h. i Quantification of ISRE-Luc activity in 2fTGH-ISRE-Luc cells transfected with a negative control siRNA (siCtrl) or UBR5 siRNA for 48 h, then poly (I:C) for 12 h. j Measurement of the Luc activity in HEK293T cells transfected with a pcDNA3.1 vector or UBR5 expression plasmid, together with an IFNB1 promoter-driven firefly luciferase (Luc) and an ACTIN promoter-driven renilla Luc plasmid (internal control) for 24 h. The cells were then transfected with poly (I:C) for 12 h. k Quantification of the IFNB1 mRNA in HEK293T cells transfected with a vector or UBR5 expression plasmid for 24 h, then poly (I:C) for 12 h. l Immunoblots of UBR5 protein in mouse primary embryonic fibroblasts (MEF) or bone marrow-derived macrophages. Quantification of ( m ) the Ifnb1 mRNA in MEFs, secreted IFN-β protein in ( n ) MEFs and ( o ) macrophages transfected with poly (I:C). Quantification of the ( p ) Ifnb1 mRNA levels, q secreted IFN-β protein in MEFs transfected with 5-ppp hpRNA (RIG-I agonist). Ubr5 iKO : Ubr5 inducible knockout by Tamoxifen. Data presented in b – d , g – i : mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, n = 3 biological independent experiments; for b : **** p < 0.0001, *** p = 0.0003, * p = 0.0305 vs WT; for c : **** p < 0.0001 vs WT; for d : *** p = 0.0003, ** p = 0.0022 vs WT; for g : *** p = 0.0001, **** p < 0.0001 vs WT; for h : **** p < 0.0001 vs WT; for i : **** p < 0.0001, *** p = 0.0005 vs siCtrl. Mult i plicity adjusted p values are p resented. Data presented in e , f , j , k , m – q : mean ± S.E.M, two-tailed student’s t test; for e : *** p = 0.0007, **** p < 0.0001, n = 4 biological independent experiments; for f : *** p = 0.0002, * p = 0.012; for j : ** p = 0.0015, ** p = 0.0018 in sequence; for k : ** p = 0.0014, ** p = 0.0084 in sequence; for m : * p = 0.0224, **** p < 0.0001; for n : ** p = 0.0024, * p = 0.0407; for o : * p = 0.0478, * p = 0.0129, *** p = 0.0002 in sequence; for p : *** p = 0.0005, *** p = 0.0001 in sequence; for q : * p = 0.0263, * p = 0.0138 in sequence; n = 3 biological independent experiments in f , j , k , m – q . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Bioassay, Negative Control, Plasmid Preparation, Expressing, Luciferase, Control, Derivative Assay, Knock-Out, Two Tailed Test, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a The immunoblots of UBR5 in various tissues of age- and sex-matched littermates. Black triangle indicates the right band size. The survival curves of littermates infected with ( b ) 100 or ( c ) 1,000 plaque forming units (PFU) of EMCV intraperitoneally. In b , n = 16 for Ubr5 WT and 17 for Ubr5 iKO , p = 0.038 (Log-Rank test); in c , n = 14 for Ubr5 WT and 10 for Ubr5 iKO , p = 0.030 (Log-Rank test). Quantification of ( d ) EMCV RNA in the whole blood cells by qRT-PCR, e viremia and ( f ) viral loads in hearts by a plaque forming assay, in the mice infected with 100 PFU of EMCV. In d – f , n = 6 mice, mean ± S.E.M., two-tailed, unpaired non–parametric Mann–Whitney U test; * p = 0.0411 ( d ), * p = 0.0152 ( e ), * p = 0.0411 ( f ). g , h Quantification of the serum type I IFN and cytokine concentrations by ELISA in the mice infected with 1 × 10 7 PFU of EMCV intravenously. n = 7 mice/group, mean ± S.E.M., two-tailed Student’s t test; *** p = 0.0009 for IFN-β, * p = 0.0221 for IFN-α ( g ); * p = 0.0259 for TNF-α, ** p = 0.0055 for CXCL10, ** p = 0.0034 for MIP−1α, ** p = 0.0049 for MIP−1β ( h ). i The survival curves of age- and sex-matched littermates infected with 1 × 10 7 PFU of VSV intravenously. n = 11 mice/group, p = 0.03 (Log-Rank test). j The arbitrary morbidity score of VSV-infected animals. n = 6 mice/group, mean ± S.E.M., two-tailed Student’s t test; * p = 0.0438, * p = 0.0438 in sequence. Adjusted p values are presented. All the mice used in this study were 8 weeks old. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Infection, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: The immunoblots of indicated proteins in ( a ) mouse primary MEFs, macrophages and various human cell lines, ( b ) the tissues of age- and sex-matched littermates. The immunoblots of indicated proteins in HEK293T cells ( c ) transfected with poly (I:C)/without (Mock) for 12 h, and ( d ) infected with VSV at a multiplicity of infection (MOI) of 0.5. e The immunoblots of indicated proteins in UBR5 −/− HEK293T cells transfected with a UBR5 expression or vector plasmid for 24 h and then poly (I:C) (+) for 12 h. The bar chart indicates the ratios of MDA5/RIG-I band density to Actin. f The immunoblots of indicated proteins in WT HEK293T cells transfected with a vector, wild-type UBR5, or catalytic mutant C2768A plasmid. The bar chart indicates the ratios of MDA5/RIG-I band density to Actin. n = 2 biologically independent experiments. qRT-PCR quantification of the IFIH1 / DDX58 (gene symbol for MDA5/RIG-I) mRNA levels in ( g ) HEK293T cells, and ( h ) MEFs transfected with poly (I:C). The data are representative of three independent experiments ( a , c , d ) or tissues from two mice ( b ) with similar results. The data shown in ( e ) are from one representative experiment of n = 3 biological independent experiments, mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, *** p = 0.0008, ** p = 0.0036 for MDA5; *** p = 0.0009, ** p = 0.0023 for RIG-I. Multiplicity adjusted p values are presented. Data shown in g , h : mean ± S.E.M, two-tailed Student’s t test, n = 3 biologically independent experiments; *** p = 0.0004, ** p = 0.0028 in g ; for h : * p = 0.0493, * p = 0.0458, ** p = 0.0017 in sequence for Ifih1 ; * p = 0.0286, ** p = 0.0086, * p = 0.0472 in sequence for Ddx58 . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Transfection, Infection, Expressing, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Two Tailed Test, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a The immunoblots of indicated proteins in 2fTGH cells transfected with poly (I:C) or infected with VSV-GFP at a MOI of 0.5. b The fluorescent images of VSV-GFP in 2fTGH cells at 12 h p.i . Scale bar: 50 µM. Quantification of the ( c ) IFIH1 / DDX58 (gene symbol for MDA5/RIG-I) and ( d ) IFNB1 mRNA levels by qRT-PCR in 2fTGH cells infected with VSV for 12 h. Data shown in c and d are presented as mean ± S.E.M, two-tailed Student’s t test, n = 3 biologically independent experiments; * p = 0.0492, * p = 0.0189 in sequence for IFIH1 , * p = 0.0286, * p = 0.0483 in sequence for DDX58 in c ; * p = 0.0489 in d . Adjusted p values are presented. e Scatter plot showing the 49 proteins identified by FLAG-UBR5- immunoprecipitated (IP)-mass spectrometer (MS) analysis. The 17 proteins validated at Harmonizome database are labeled. Positive proteins: log 2 (FC of A.P.I) > 1, two-tailed Student’s t test with Benjamini–Hochberg, p < 0.05, FC of A.P.I: fold change of Average Precursor Intensity. f , g FLAG-UBR5 co-immunoprecipitated (IP) with endogenous TRIM28, and vice versa. FLAG-UBR5/TRIM28 or vector plasmid was expressed in HEK293T cells, and immunoprecipitated with an anti-FLAG antibody. The indicated proteins were immunoblotted (IB) with specific antibodies. WCL: whole cell lysate. The immunoblots of indicated proteins in HEK293T cells transfected with ( h ) a negative siRNA (Ctrl) or UBR5 siRNA for 48 h, ( i ) with a UBR5 express i on or vector plasmid for 24 h. The data are representative of two independent experiments with similar results ( a , f – i ). Source data a re provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Transfection, Infection, Quantitative RT-PCR, Two Tailed Test, Sequencing, Immunoprecipitation, Mass Spectrometry, Labeling, Plasmid Preparation

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28, His-UBC9 and HA- or Myc -SUMO plasmids for 24 h, then without (Mock) or with poly (I:C) for 12 h. FLAG-TRIM28 was immunoprecipitated (IP) with an anti-FLAG antibody, and the IP and whole cell lysate (WCL) were immuno-blotted (IB) for the indicated proteins with specific antibodies. b WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28, HA-tagged WT, K48 or K63-only ubiquitin (Ub) for 24 h. The IP and IB was performed as above. c WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28 or vector for 24 h, then with poly (I:C) for 12 h. The IP and IB was carried out as above. The bar chart in a – c indicates the ratios of the indicated protein band density. n = 2 biologically independent experiments. d WT and UBR5 −/− HEK293T cells were infected with VSV at a MOI of 0.5. Endogenous TRIM28 was immunoprecipitated with an anti-TRIM28 antibody. e The bar chart indicates the ratios of the indicated protein band density in d . n = 2 biologically independent experiments. f The method to identify ubiquitinated sites within TRIM28. g The method for generating K507R and K779R mutants of TRIM28. h HEK293T cells were transfected with FLAG-TRIM28 (WT, K507R, K779R), GFP-UBR5, GFP-UBR5-C2758A mutant and HA-K63Ub plasmids for 24 h, then FLAG-TRIM28 was immunoprecipitated with an anti-FLAG antibody, and the IP and WCL were immunoblotted for the indicated proteins with specific antibodies. I – k WT and UBR5 −/− HEK293T cells were infected with VSV at a MOI of 0.5 for 8 h. Chromatin immunoprecipitation (ChIP) was performed with an anti-TRIM28 antibody. i The TRIM28-bound RLR promoter DNA was quantified by qPCR and normalized to its input. Bar: mean ± S.E.M, two-tailed Student’s t test, n = 4 biologically independent samples, *** p = 0.0006, ** p = 0.0094 for IFIH1 ; ** p = 0.0029; * p = 0.0382. Adjusted p values are presented. j ChIP-seq analysis and genomic annotation of TRIM28-bound sites in infected cells. UTR: untranslated regions. k Fold enrichment inTRIM28-bound promoter regions of select ISGs. The P -value was generated in Peak calling statistics using a Poisson distribution with local lambda estimate. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Transfection, Immunoprecipitation, Ubiquitin Proteomics, Plasmid Preparation, Infection, Mutagenesis, Chromatin Immunoprecipitation, Two Tailed Test, ChIP-sequencing, Generated

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a , b Volcano plots of differentially expressed genes (DEGs) in UBR5 −/− versus WT cells with/or without poly (I:C)-stimulation. Red and blue represent significant DEGs of upregulated (log 2 FC ≥ 1, p < 0.05) and downregulated (log 2 FC ≤ −1, p < 0.05), res p ectively. DESeq2 was used to perform a comparison of gene expression between defined groups, the Wald test was used to generate log 2 FC and p -values adjusted with the Benjamini–Hochberg. c GSEA plot of a significant gene set associated with RLR pathway and ISGs, p values were calculated by one-way ANOVA with Tukey’s post hoc comparison. d Top KEGG and Reactome pathways enriched from downregulated DEGs in UBR5 −/− versus WT cells upon poly (I:C)-stimulation ( p < 0.05, right-tailed Fisher’s exact t test with Benjamini & Hochberg). e Venn diagram revealing 42 genes overlapping between the DEGs upregulated in TRIM28 −/− and downregulated in UBR5 −/− cells. f Top Reactome pathways enriched from upregulated DEGs in TRIM28 −/− versus WT cells upon poly (I:C)-stimulation. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Comparison, Gene Expression

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a Immunoblots of UBR5 protein in wild type (WT) and three UBR5 −/− (made with unique guide RNA) in 2fTGH-ISRE-Luc cells. Quantification of the ( b ) intracellular ISRE-Luc activity, c secreted IFN-β protein by ELISA, d cellular IFNB1 mRNA levels in 2fTGH-ISRE-Luc cells transfected with poly (I:C) for 12 h. Quantification of the ( e ) intracellular ISRE-Luc activity, f secreted IFN-β protein, g cellular IFNB1 mRNA, h secreted type I IFNs (Bioassay) in WT, UBR5 −/− (made with gRNA #1) and IFIH1 −/− (gene symbol for MDA5) HEK293T cells transfected with poly (I:C) for 12 h. i Quantification of ISRE-Luc activity in 2fTGH-ISRE-Luc cells transfected with a negative control siRNA (siCtrl) or UBR5 siRNA for 48 h, then poly (I:C) for 12 h. j Measurement of the Luc activity in HEK293T cells transfected with a pcDNA3.1 vector or UBR5 expression plasmid, together with an IFNB1 promoter-driven firefly luciferase (Luc) and an ACTIN promoter-driven renilla Luc plasmid (internal control) for 24 h. The cells were then transfected with poly (I:C) for 12 h. k Quantification of the IFNB1 mRNA in HEK293T cells transfected with a vector or UBR5 expression plasmid for 24 h, then poly (I:C) for 12 h. l Immunoblots of UBR5 protein in mouse primary embryonic fibroblasts (MEF) or bone marrow-derived macrophages. Quantification of ( m ) the Ifnb1 mRNA in MEFs, secreted IFN-β protein in ( n ) MEFs and ( o ) macrophages transfected with poly (I:C). Quantification of the ( p ) Ifnb1 mRNA levels, q secreted IFN-β protein in MEFs transfected with 5-ppp hpRNA (RIG-I agonist). Ubr5 iKO : Ubr5 inducible knockout by Tamoxifen. Data presented in b – d , g – i : mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, n = 3 biological independent experiments; for b : **** p < 0.0001, *** p = 0.0003, * p = 0.0305 vs WT; for c : **** p < 0.0001 vs WT; for d : *** p = 0.0003, ** p = 0.0022 vs WT; for g : *** p = 0.0001, **** p < 0.0001 vs WT; for h : **** p < 0.0001 vs WT; for i : **** p < 0.0001, *** p = 0.0005 vs siCtrl. Mult i plicity adjusted p values are p resented. Data presented in e , f , j , k , m – q : mean ± S.E.M, two-tailed student’s t test; for e : *** p = 0.0007, **** p < 0.0001, n = 4 biological independent experiments; for f : *** p = 0.0002, * p = 0.012; for j : ** p = 0.0015, ** p = 0.0018 in sequence; for k : ** p = 0.0014, ** p = 0.0084 in sequence; for m : * p = 0.0224, **** p < 0.0001; for n : ** p = 0.0024, * p = 0.0407; for o : * p = 0.0478, * p = 0.0129, *** p = 0.0002 in sequence; for p : *** p = 0.0005, *** p = 0.0001 in sequence; for q : * p = 0.0263, * p = 0.0138 in sequence; n = 3 biological independent experiments in f , j , k , m – q . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Bioassay, Negative Control, Plasmid Preparation, Expressing, Luciferase, Control, Derivative Assay, Knock-Out, Two Tailed Test, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a The immunoblots of UBR5 in various tissues of age- and sex-matched littermates. Black triangle indicates the right band size. The survival curves of littermates infected with ( b ) 100 or ( c ) 1,000 plaque forming units (PFU) of EMCV intraperitoneally. In b , n = 16 for Ubr5 WT and 17 for Ubr5 iKO , p = 0.038 (Log-Rank test); in c , n = 14 for Ubr5 WT and 10 for Ubr5 iKO , p = 0.030 (Log-Rank test). Quantification of ( d ) EMCV RNA in the whole blood cells by qRT-PCR, e viremia and ( f ) viral loads in hearts by a plaque forming assay, in the mice infected with 100 PFU of EMCV. In d – f , n = 6 mice, mean ± S.E.M., two-tailed, unpaired non–parametric Mann–Whitney U test; * p = 0.0411 ( d ), * p = 0.0152 ( e ), * p = 0.0411 ( f ). g , h Quantification of the serum type I IFN and cytokine concentrations by ELISA in the mice infected with 1 × 10 7 PFU of EMCV intravenously. n = 7 mice/group, mean ± S.E.M., two-tailed Student’s t test; *** p = 0.0009 for IFN-β, * p = 0.0221 for IFN-α ( g ); * p = 0.0259 for TNF-α, ** p = 0.0055 for CXCL10, ** p = 0.0034 for MIP−1α, ** p = 0.0049 for MIP−1β ( h ). i The survival curves of age- and sex-matched littermates infected with 1 × 10 7 PFU of VSV intravenously. n = 11 mice/group, p = 0.03 (Log-Rank test). j The arbitrary morbidity score of VSV-infected animals. n = 6 mice/group, mean ± S.E.M., two-tailed Student’s t test; * p = 0.0438, * p = 0.0438 in sequence. Adjusted p values are presented. All the mice used in this study were 8 weeks old. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Infection, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: The immunoblots of indicated proteins in ( a ) mouse primary MEFs, macrophages and various human cell lines, ( b ) the tissues of age- and sex-matched littermates. The immunoblots of indicated proteins in HEK293T cells ( c ) transfected with poly (I:C)/without (Mock) for 12 h, and ( d ) infected with VSV at a multiplicity of infection (MOI) of 0.5. e The immunoblots of indicated proteins in UBR5 −/− HEK293T cells transfected with a UBR5 expression or vector plasmid for 24 h and then poly (I:C) (+) for 12 h. The bar chart indicates the ratios of MDA5/RIG-I band density to Actin. f The immunoblots of indicated proteins in WT HEK293T cells transfected with a vector, wild-type UBR5, or catalytic mutant C2768A plasmid. The bar chart indicates the ratios of MDA5/RIG-I band density to Actin. n = 2 biologically independent experiments. qRT-PCR quantification of the IFIH1 / DDX58 (gene symbol for MDA5/RIG-I) mRNA levels in ( g ) HEK293T cells, and ( h ) MEFs transfected with poly (I:C). The data are representative of three independent experiments ( a , c , d ) or tissues from two mice ( b ) with similar results. The data shown in ( e ) are from one representative experiment of n = 3 biological independent experiments, mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, *** p = 0.0008, ** p = 0.0036 for MDA5; *** p = 0.0009, ** p = 0.0023 for RIG-I. Multiplicity adjusted p values are presented. Data shown in g , h : mean ± S.E.M, two-tailed Student’s t test, n = 3 biologically independent experiments; *** p = 0.0004, ** p = 0.0028 in g ; for h : * p = 0.0493, * p = 0.0458, ** p = 0.0017 in sequence for Ifih1 ; * p = 0.0286, ** p = 0.0086, * p = 0.0472 in sequence for Ddx58 . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Transfection, Infection, Expressing, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Two Tailed Test, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a The immunoblots of indicated proteins in 2fTGH cells transfected with poly (I:C) or infected with VSV-GFP at a MOI of 0.5. b The fluorescent images of VSV-GFP in 2fTGH cells at 12 h p.i . Scale bar: 50 µM. Quantification of the ( c ) IFIH1 / DDX58 (gene symbol for MDA5/RIG-I) and ( d ) IFNB1 mRNA levels by qRT-PCR in 2fTGH cells infected with VSV for 12 h. Data shown in c and d are presented as mean ± S.E.M, two-tailed Student’s t test, n = 3 biologically independent experiments; * p = 0.0492, * p = 0.0189 in sequence for IFIH1 , * p = 0.0286, * p = 0.0483 in sequence for DDX58 in c ; * p = 0.0489 in d . Adjusted p values are presented. e Scatter plot showing the 49 proteins identified by FLAG-UBR5- immunoprecipitated (IP)-mass spectrometer (MS) analysis. The 17 proteins validated at Harmonizome database are labeled. Positive proteins: log 2 (FC of A.P.I) > 1, two-tailed Student’s t test with Benjamini–Hochberg, p < 0.05, FC of A.P.I: fold change of Average Precursor Intensity. f , g FLAG-UBR5 co-immunoprecipitated (IP) with endogenous TRIM28, and vice versa. FLAG-UBR5/TRIM28 or vector plasmid was expressed in HEK293T cells, and immunoprecipitated with an anti-FLAG antibody. The indicated proteins were immunoblotted (IB) with specific antibodies. WCL: whole cell lysate. The immunoblots of indicated proteins in HEK293T cells transfected with ( h ) a negative siRNA (Ctrl) or UBR5 siRNA for 48 h, ( i ) with a UBR5 express i on or vector plasmid for 24 h. The data are representative of two independent experiments with similar results ( a , f – i ). Source data a re provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Transfection, Infection, Quantitative RT-PCR, Two Tailed Test, Sequencing, Immunoprecipitation, Mass Spectrometry, Labeling, Plasmid Preparation

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28, His-UBC9 and HA- or Myc -SUMO plasmids for 24 h, then without (Mock) or with poly (I:C) for 12 h. FLAG-TRIM28 was immunoprecipitated (IP) with an anti-FLAG antibody, and the IP and whole cell lysate (WCL) were immuno-blotted (IB) for the indicated proteins with specific antibodies. b WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28, HA-tagged WT, K48 or K63-only ubiquitin (Ub) for 24 h. The IP and IB was performed as above. c WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28 or vector for 24 h, then with poly (I:C) for 12 h. The IP and IB was carried out as above. The bar chart in a – c indicates the ratios of the indicated protein band density. n = 2 biologically independent experiments. d WT and UBR5 −/− HEK293T cells were infected with VSV at a MOI of 0.5. Endogenous TRIM28 was immunoprecipitated with an anti-TRIM28 antibody. e The bar chart indicates the ratios of the indicated protein band density in d . n = 2 biologically independent experiments. f The method to identify ubiquitinated sites within TRIM28. g The method for generating K507R and K779R mutants of TRIM28. h HEK293T cells were transfected with FLAG-TRIM28 (WT, K507R, K779R), GFP-UBR5, GFP-UBR5-C2758A mutant and HA-K63Ub plasmids for 24 h, then FLAG-TRIM28 was immunoprecipitated with an anti-FLAG antibody, and the IP and WCL were immunoblotted for the indicated proteins with specific antibodies. I – k WT and UBR5 −/− HEK293T cells were infected with VSV at a MOI of 0.5 for 8 h. Chromatin immunoprecipitation (ChIP) was performed with an anti-TRIM28 antibody. i The TRIM28-bound RLR promoter DNA was quantified by qPCR and normalized to its input. Bar: mean ± S.E.M, two-tailed Student’s t test, n = 4 biologically independent samples, *** p = 0.0006, ** p = 0.0094 for IFIH1 ; ** p = 0.0029; * p = 0.0382. Adjusted p values are presented. j ChIP-seq analysis and genomic annotation of TRIM28-bound sites in infected cells. UTR: untranslated regions. k Fold enrichment inTRIM28-bound promoter regions of select ISGs. The P -value was generated in Peak calling statistics using a Poisson distribution with local lambda estimate. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Transfection, Immunoprecipitation, Ubiquitin Proteomics, Plasmid Preparation, Infection, Mutagenesis, Chromatin Immunoprecipitation, Two Tailed Test, ChIP-sequencing, Generated

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a , b Volcano plots of differentially expressed genes (DEGs) in UBR5 −/− versus WT cells with/or without poly (I:C)-stimulation. Red and blue represent significant DEGs of upregulated (log 2 FC ≥ 1, p < 0.05) and downregulated (log 2 FC ≤ −1, p < 0.05), res p ectively. DESeq2 was used to perform a comparison of gene expression between defined groups, the Wald test was used to generate log 2 FC and p -values adjusted with the Benjamini–Hochberg. c GSEA plot of a significant gene set associated with RLR pathway and ISGs, p values were calculated by one-way ANOVA with Tukey’s post hoc comparison. d Top KEGG and Reactome pathways enriched from downregulated DEGs in UBR5 −/− versus WT cells upon poly (I:C)-stimulation ( p < 0.05, right-tailed Fisher’s exact t test with Benjamini & Hochberg). e Venn diagram revealing 42 genes overlapping between the DEGs upregulated in TRIM28 −/− and downregulated in UBR5 −/− cells. f Top Reactome pathways enriched from upregulated DEGs in TRIM28 −/− versus WT cells upon poly (I:C)-stimulation. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Comparison, Gene Expression