pcmv tag2b ubr5 c2768a  (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: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 2 Inducible Ubr5 expression knockdown suppresses post-surgery lung metastasis. (A) Schematic for removing primary tumor and knocking down Ubr5 expression with daily Dox addition (100 mg/kg) by gavage post-surgery in mice. (B, C) Representative images for lung nodules stained with India ink (B) Lung nodules in tumor-bearing are quantified (C). (D, E) The 6-thioguanine clonogenicity assay of Tet-scr and Tet-shUbr5 tumors (on D35) for lung metastasis is conducted. Images of representative experiment are depicted (D) and colonies are quantified (E). (F) Overall survival rates of the mice (Tet-scr, n = 8; Tet-scr(+Dox), n = 6; Tet-shUbr5, n = 5; Tet-shUbr5(+Dox), n = 5). (G) UBR5 protein expression in hUBR5- and C2768A-reconstituted 4T1/Ubr5/ cells is evaluated in Western blotting. (H) Representative result of E2 discharge assay for UBR5 and C2768A. (I) Quantitation of the discharged E2-Ub by UBR5 and C2768A. (J) A total of 5 105 Tet-scr, Tet-shUbr5, Ubr5/ + hUBR5 or Ubr5/ + C2768A cells are injected into 6- to 8-week-old female BALB/c mice (with 8, 8, 8 and 16 mice, respectively, for the four cell lines). Tumor volume is measured on D20. (K) The metastatic cells in the lung are enumerated by the 6-thiogaunine assay. (L) After removing the primary tumor, the mice with similar primary tumor volumes are selected from all groups and administrated with Dox daily by gavage (100 mg/kg). On D30, the mice are sacrificed. Lung metastatic nodules are stained with Indian ink and quantified (Tet-scr, n = 5; Tet-shUbr5, n = 5; Ubr5/ + hUBR5, n = 5, Ubr5/ + C2768A, n = 4). Data represents mean ± SEM; *P < .05; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Expressing, Knockdown, Staining, Western Blot, Quantitation Assay, Injection

Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 1 Limited primary tumor growth and less lung metastasis in mice carrying inducible Ubr5-knockdown 4T1 cells. (A) Schematic diagram of the experimental design. 5 105 4T1-GFP, 4T1-Tet-shUbr5 cells are injected into the mammary gland of female BALB/c mice and on D3, the mice are treated with 100 mg/kg Dox by gavage daily. On day 27, mice are sacrificed, and tumors are dissected. (B) 4T1 tumor growth curve. (C) Tumor weight is measured. (D) Ubr5 mRNA expression of mice bearing tumor is detected by real-time quantitative PCR. (E). Protein level of UBR5 in tumor analyzed by Western blot. (F) Representative UBR5 IHC staining of tumor from mice bearing 4T1/GFP, 4T1/Tet-shUbr5 cells. (G) Statistical analysis of UBR5 immunostaining IRS score. (H) Palpable metastatic nodules on lung surfaces are enumerated. Data represents mean ± SEM; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Knockdown, Injection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Immunostaining

Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 2 Inducible Ubr5 expression knockdown suppresses post-surgery lung metastasis. (A) Schematic for removing primary tumor and knocking down Ubr5 expression with daily Dox addition (100 mg/kg) by gavage post-surgery in mice. (B, C) Representative images for lung nodules stained with India ink (B) Lung nodules in tumor-bearing are quantified (C). (D, E) The 6-thioguanine clonogenicity assay of Tet-scr and Tet-shUbr5 tumors (on D35) for lung metastasis is conducted. Images of representative experiment are depicted (D) and colonies are quantified (E). (F) Overall survival rates of the mice (Tet-scr, n = 8; Tet-scr(+Dox), n = 6; Tet-shUbr5, n = 5; Tet-shUbr5(+Dox), n = 5). (G) UBR5 protein expression in hUBR5- and C2768A-reconstituted 4T1/Ubr5/ cells is evaluated in Western blotting. (H) Representative result of E2 discharge assay for UBR5 and C2768A. (I) Quantitation of the discharged E2-Ub by UBR5 and C2768A. (J) A total of 5 105 Tet-scr, Tet-shUbr5, Ubr5/ + hUBR5 or Ubr5/ + C2768A cells are injected into 6- to 8-week-old female BALB/c mice (with 8, 8, 8 and 16 mice, respectively, for the four cell lines). Tumor volume is measured on D20. (K) The metastatic cells in the lung are enumerated by the 6-thiogaunine assay. (L) After removing the primary tumor, the mice with similar primary tumor volumes are selected from all groups and administrated with Dox daily by gavage (100 mg/kg). On D30, the mice are sacrificed. Lung metastatic nodules are stained with Indian ink and quantified (Tet-scr, n = 5; Tet-shUbr5, n = 5; Ubr5/ + hUBR5, n = 5, Ubr5/ + C2768A, n = 4). Data represents mean ± SEM; *P < .05; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Expressing, Knockdown, Staining, Western Blot, Quantitation Assay, Injection

Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 4 UBR5 suppresses apoptosis and promotes lung metastasis via targeting CDC73. (A) UBR5 and CDC73 protein expression in 4T1 cells with Dox-induced shUbr5 and shCdc73 expression. (B) Representative FACS images of Dox-treated Tet-scr, Tet-shUbr5, Tet-shCdc73 and Tet-shUbr5-shCdc73 cells exposed to Dovitinib (1 μm) for 24 h. (C) Quantitation of apoptotic cells in b. (D) Apoptosis in lung sections of mice bearing Dox-treated Tet-scr, Tet-shUbr5, Tet-shCdc73 or Tet-shUbr5-shCdc73 tumors, respectively, is evaluated by TUNEL staining on D35 (n = 3 mice per group), Scale bar: 100 μm. (E) Representative IHC staining for Ki67 expression images in the lung with quantifications. (F) Lung metastatic nodules in tumor-bearing are stained with India ink and quantified (5 mice/group). (G) Overall survival rates of mice carrying Dox- treated Tet-scr (n = 7), Tet-shUbr5 (n = 5), Tet-shCdc73 (n = 6) or Tet-shUbr5-shCdc73 (n = 8) tumors. Data represents mean ± SEM; *P < .05; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Expressing, Quantitation Assay, TUNEL Assay, Staining, Immunohistochemistry

Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 6 Effects of human TNBC-expressed UBR5 on tumor growth and lung metastases. (A) IHC staining of UBR5 and CDC73 proteins in primary tumors from TNBC patients (n = 70 samples). Scale bars: 200 μm. (B) Correlation between tumor UBR5 and CDC73 expression levels in human TNBC patients. (C) mRNA levels of CDC73 in primary breast cancer tissues (n = 168) and metastatic sites (n = 52). (D) Metastatic nodules in the lung and liver of tumor-bearing mice are quantified on D45. (E) Representative IHC staining of UBR5 and CDC73 in lung metastatic nodules. Scale bar: 100 μm. (F) UBR5 and CDC73 protein expression in MDA-MB-231-Vector, MDA-MB-231-shUBR5 or MDA-MB- 231-shUBR5-shCDC73 cell lines. (G) Tumor growth rates of NSG mice. (H) Metastatic nodules in the lung and liver of tumor-bearing mice are quantified on D38. Data represents mean ± SEM; *P < .05; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Immunohistochemistry, Expressing, Plasmid Preparation

Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 2 Inducible Ubr5 expression knockdown suppresses post-surgery lung metastasis. (A) Schematic for removing primary tumor and knocking down Ubr5 expression with daily Dox addition (100 mg/kg) by gavage post-surgery in mice. (B, C) Representative images for lung nodules stained with India ink (B) Lung nodules in tumor-bearing are quantified (C). (D, E) The 6-thioguanine clonogenicity assay of Tet-scr and Tet-shUbr5 tumors (on D35) for lung metastasis is conducted. Images of representative experiment are depicted (D) and colonies are quantified (E). (F) Overall survival rates of the mice (Tet-scr, n = 8; Tet-scr(+Dox), n = 6; Tet-shUbr5, n = 5; Tet-shUbr5(+Dox), n = 5). (G) UBR5 protein expression in hUBR5- and C2768A-reconstituted 4T1/Ubr5/ cells is evaluated in Western blotting. (H) Representative result of E2 discharge assay for UBR5 and C2768A. (I) Quantitation of the discharged E2-Ub by UBR5 and C2768A. (J) A total of 5 105 Tet-scr, Tet-shUbr5, Ubr5/ + hUBR5 or Ubr5/ + C2768A cells are injected into 6- to 8-week-old female BALB/c mice (with 8, 8, 8 and 16 mice, respectively, for the four cell lines). Tumor volume is measured on D20. (K) The metastatic cells in the lung are enumerated by the 6-thiogaunine assay. (L) After removing the primary tumor, the mice with similar primary tumor volumes are selected from all groups and administrated with Dox daily by gavage (100 mg/kg). On D30, the mice are sacrificed. Lung metastatic nodules are stained with Indian ink and quantified (Tet-scr, n = 5; Tet-shUbr5, n = 5; Ubr5/ + hUBR5, n = 5, Ubr5/ + C2768A, n = 4). Data represents mean ± SEM; *P < .05; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Expressing, Knockdown, Staining, Western Blot, Quantitation Assay, Injection

Journal: Scientific reports

Article Title: The E3 ubiquitin ligase UBR5 interacts with TTC7A and may be associated with very early onset inflammatory bowel disease.

doi: 10.1038/s41598-020-73482-6

Figure Lengend Snippet: Figure 5. UBR5 L1405V mutant shows significantly reduced binding to TTC7A in HEK 293 T cells. (A) TTC7A co-IPs with differential binding affinities to UBR5 mutants. UBR5 plasmids were tagged with FLAG and the His tag removed. Lysate and IP samples were derived from the same experiment but ran on different blots. (B) UBR5 L1405V and C2768A show reduced and increased binding to TTC7A, respectively. Densitometry of western blot experiments from (A) were quantified and the values from all samples were made relative to the samples transfected with TTC7A and UBR5. Error bars indicate SD. *Denotes p < 0.05, ns = non-significant (student’s t-test). n = 4. Full length blots for (A) are presented in Supplementary Figure 5.

Article Snippet: Plasmid constructs. pCMV-Tag2B EDD and C2768A were a gift from Darren Saunders & Charles Watts (Addgene plasmid # 37188 and 37189 respectively)43.

Techniques: Mutagenesis, Binding Assay, Derivative Assay, Western Blot, Transfection