ubr5 antibody  (Bethyl)

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: The anti-UBR5 antibody (Cat# A300-573A, 1:1000) was purchased from Bethyl Laboratories, Inc (Montgomery, TX, United States).

Techniques: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control, Plasmid Preparation, Expressing, Luciferase, 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: The anti-UBR5 antibody (Cat# A300-573A, 1:1000) was purchased from Bethyl Laboratories, Inc (Montgomery, TX, 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: The anti-UBR5 antibody (Cat# A300-573A, 1:1000) was purchased from Bethyl Laboratories, Inc (Montgomery, TX, 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: The anti-UBR5 antibody (Cat# A300-573A, 1:1000) was purchased from Bethyl Laboratories, Inc (Montgomery, TX, 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: The anti-UBR5 antibody (Cat# A300-573A, 1:1000) was purchased from Bethyl Laboratories, Inc (Montgomery, TX, United States).

Techniques: Transfection, Immunoprecipitation, 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: The anti-UBR5 antibody (Cat# A300-573A, 1:1000) was purchased from Bethyl Laboratories, Inc (Montgomery, TX, United States).

Techniques: Comparison, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Quantitative interactome proteomics identifies a proteostasis network for GABA A receptors

doi: 10.1016/j.jbc.2022.102423

Figure Lengend Snippet: Cellular model of the proteostasis network for GABA A receptors. Interactors that were identified from our SILAC analysis are included in each step. Interactors that are expected to play a role in the folding and assembly of GABA A receptors are colored in green ; those in their degradation, in orange ; and those in their transport, in purple . The GABA A receptor subunit is cotranslationally translocated to the ER membrane (state 1). The OST complex, including DDOST, RPN1, and RPN2, installs N -linked glycans to the subunit (state 1 to state 2). The glycoprotein is trimmed by ER α-glucosidases, such as GANAB (state 2 to state 3), and subjected to calnexin (CANX) folding cycles in a glycan-dependent manner (state 3 to state 4). In parallel, heat shock proteins and their cochaperones facilitate the folding of the subunit both in the ER lumen (HspA5, DNAJB11, and P4HB) and in the cytosol (HspA1A, HspA6, HspA8, DNAJA1, DNAJA2, and DNAJA3) (state 2 to state 4). After coordinated folding efforts (state 4), the subunit faces three possible routes. First , if folded into the native state, the subunit assembles with other subunits to form a heteropentamer with the assistance from assembly factors (state 4 to state 5). The assembled receptors engage the COPII machinery, including SEC16A, for anterograde trafficking to the Golgi and to the plasma membrane (state 5 to state 6 and state 7). The COPI machinery, including COPA, COPG2, aids the recycle of the subunits from the Golgi back to the ER (state 6 to state 5). Second , non-native subunits are reglucosylated by UGGT1 for additional folding attempts (state 4 to state 3). Third , terminally misfolded subunits undergo ERAD pathway, being recognized, ubiquitinated, dislocated, and targeted to the proteasome for degradation (state 4 to state 8 and state 9). Ubiquitin E3 ligases (UBR5, UBE3C, TRIM21, LTN1, and HUWE1) and other ERAD factors (Hsp90B1, Hsp90AB1, AHSA1, SEL1L, VCP, and ERLIN2) potentially play a critical role in this process. Aggregated subunits could utilize the lysosome-related degradation pathway (state 4 to state 10). ER, endoplasmic reticulum; ERAD, ER-associated degradation; GABA A , gamma-aminobutyric acid type A; OST, oligosaccharyltransferase; SILAC, stable isotope labeling by amino acids in cell culture; UGGT1, UDP-glucose glycoprotein glucosyltransferase 1.

Article Snippet: The rabbit polyclonal anti-UBR5 antibody (catalog no.: A300-573A) was obtained from Bethyl Laboratories.

Techniques: Labeling, Cell Culture

Journal:

Article Title:

doi:

Figure Lengend Snippet: A GSCs transduced with control or RNAi targeting CDH1, CUL4A, UBR5, and WWP2 were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. B GSCs transduced with WWP2 RNAi or control were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. C Cells isolated from patient tumors were subjected to scRNA-seq. Malignant cells were identified by expression-based inference of copy number variation as in Fig. . Cells are colored by expression level for WWP2. D GSC lysates were subjected to anti-SOX2 immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. E GSCs transduced with lentiviruses to stably express 6X-Histidine epitope-tagged SOX2 and FLAG epitope-tagged ubiquitin were transduced with WWP2 RNAi or control. Lysates were subjected to IP under denaturing conditions using nickel-NTA beads. IP and input samples were subjected to immunoblotting. Data were representative of three independent experiments. F GSCs transduced with WWP2 RNAi or control were subjected to ELDA as in Fig. . Data represent mean ± SEM ( n = 3, unpaired t -test, ** P < 0.01). See Source Data File for exact P values and statistical parameters.

Article Snippet: Antibodies used for this study include monoclonal rabbit anti-SOX2 (3579) (1:1000, Cell Signaling Technology), polyclonal goat anti-SOX2 (sc-17320) (2 μg antibody per 500 μg lysate protein, Santa Cruz Biotechnology), monoclonal rabbit anti-Sox2 SP76 (1:200, Cell Marque), monoclonal rabbit anti-SOX2 D9B8N (2 μg antibody per 500 μg lysate protein, Cell Signaling Technology), monoclonal mouse anti-alpha-Tubulin (1:4000, T5168) (Sigma), monoclonal mouse anti-HA.11 epitope tag (901502) (1:1000, Biolegend), anti-HA, rabbit monoclonal C29F4, (1:1000, Cell Signaling Technology), anti-Myc, mouse monoclonal, clone 4 A6, (1:1000, Millipore-Sigma), anti-Myc, mouse monoclonal, clone 9E10, (2 μg antibody per 500 μg lysate protein, Millipore-Sigma), polyclonal rabbit anti-GFP (A6455) (1:5000, Thermo Fisher), monoclonal mouse anti-GFP (A11120) (1 μg/mL lysate, Thermo Fisher), monoclonal mouse anti-TRIM26 (sc-393832) (1:200, Santa Cruz Biotechnology), monoclonal mouse anti-beta-Actin (sc-47778) (1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-FLAG M2 (F3165) (1:2000, Sigma), polyclonal rabbit anti-His-Tag (1:1000, 2365) (Cell Signaling Technology), monoclonal mouse anti-FZR1(CDH1) (ab3242) (1:500, Abcam), polyclonal rabbit anti-Cullin-4A (1:1000, C0371) (Sigma), polyclonal rabbit anti-EDD1(UBR5) (A300-573A) (1:2000, Bethyl Laboratories), polyclonal rabbit anti-WWP2 (A302-935A) (1:2000, Bethyl Laboratories), monoclonal mouse anti-Human nuclear antigen 235-1 (1:500, Novus Biological), and Normal goat IgG (NI02) (Millipore-Sigma).

Techniques: Transduction, Western Blot, Isolation, Expressing, Immunoprecipitation, Stable Transfection, FLAG-tag

Journal: BMC Cancer

Article Title: UBR-box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target

doi: 10.1186/s12885-020-07322-1

Figure Lengend Snippet: UBR5 is altered in human lung adenocarcinoma. a UBR5 is altered by mutation or increased copy number in 10% of human lung adenocarcinoma analyzed by TCGA. b Schematic of UBR5 protein. UBR5 is a 2799 amino acid protein with an N-terminal UBA (ubiquitin-associated) domain, a middle zf-Box (zinc finger UBR-box) domain and two C-terminal domains, PABP (poly [A]-binding protein) & HECT (homology to E6-AP carboxyl terminus). c A wide variability in the UBR5 protein level was observed in various lung adenocarcinoma cell lines; Tubulin was used as a loading control. The full-length films are presented in Supplementary Fig. S . d The level of UBR5 protein was found to be highly elevated in nearly all primary human lung tumor samples (T) compared to normal adjacent lung (N), N1 represents the normal adjacent to T1, N2 is the normal adjacent to T2, etc.; GAPDH was used as a house keeping gene. The full-length films are presented in Supplementary Fig. S . e Densitometric analysis of western blot films from panel 1d as ratio of UBR5/GAPDH

Article Snippet: Western blots were performed in Bolt Bis-Tris gels (#BG4120BOX, Life Technologies, Grand island, NY, USA) as per manufacturer’s protocol using antibodies from Santa Cruz, Dallas, TX, USA (GAPDH # sc47724); Bethyl, Montgomery, TX, USA (UBR5 # A300-573A, GCL1N1 # A301-843A) and Cell Signaling, Danvers, MA, USA (FLAG # 14793, DNA-PK # 4602, mTOR # 2972, RAPTOR # 2280, RICTOR # 2114, AKT # 4691, pAKT S473 # 4060).

Techniques: Mutagenesis, Ubiquitin Proteomics, Binding Assay, Control, Western Blot

Journal: BMC Cancer

Article Title: UBR-box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target

doi: 10.1186/s12885-020-07322-1

Figure Lengend Snippet: UBR5 interacts with multiple proteins. a An abbreviated list of proteins that were identified as UBR5 interacting proteins. Unique peptide count; number of distinct peptides identified by MS/MS in UBR5 immunoprecipitates (IP). Many secretory and anti-inflammatory proteins are found to be interacting with UBR5. b HEK293T cells were transiently transfected with FLAG-tagged UBR5 followed by IP by anti-FLAG antibody and Western Blot analysis. UBR5 interact with proteins involve in DNA damage and mTOR pathway. The full-length films are presented in Supplementary Fig. S

Article Snippet: Western blots were performed in Bolt Bis-Tris gels (#BG4120BOX, Life Technologies, Grand island, NY, USA) as per manufacturer’s protocol using antibodies from Santa Cruz, Dallas, TX, USA (GAPDH # sc47724); Bethyl, Montgomery, TX, USA (UBR5 # A300-573A, GCL1N1 # A301-843A) and Cell Signaling, Danvers, MA, USA (FLAG # 14793, DNA-PK # 4602, mTOR # 2972, RAPTOR # 2280, RICTOR # 2114, AKT # 4691, pAKT S473 # 4060).

Techniques: Tandem Mass Spectroscopy, Transfection, Western Blot

Journal: BMC Cancer

Article Title: UBR-box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target

doi: 10.1186/s12885-020-07322-1

Figure Lengend Snippet: UBR5 interacts with AKT and regulate its activity. a HEK293T cells were transiently transfected with FLAG-tagged UBR5 followed by IP by anti-FLAG antibody and Western Blot analysis. The full-length films are presented in Supplementary Fig. S [Panel 1–3]. b A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5. The full-length films are presented in Supplementary Fig. S [Panel 4–5]. c Lung adenocarcinoma cell lines which are stably expressing Cas9 were transiently transfected with synthetic gRNAs against UBR5. The full-length films are presented in Supplementary Fig. S

Article Snippet: Western blots were performed in Bolt Bis-Tris gels (#BG4120BOX, Life Technologies, Grand island, NY, USA) as per manufacturer’s protocol using antibodies from Santa Cruz, Dallas, TX, USA (GAPDH # sc47724); Bethyl, Montgomery, TX, USA (UBR5 # A300-573A, GCL1N1 # A301-843A) and Cell Signaling, Danvers, MA, USA (FLAG # 14793, DNA-PK # 4602, mTOR # 2972, RAPTOR # 2280, RICTOR # 2114, AKT # 4691, pAKT S473 # 4060).

Techniques: Activity Assay, Transfection, Western Blot, Infection, shRNA, Construct, Stable Transfection, Expressing

Journal: BMC Cancer

Article Title: UBR-box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target

doi: 10.1186/s12885-020-07322-1

Figure Lengend Snippet: UBR5 deficient A549 cells show decreased cell viability and clonogenic potential. a A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5 and were cultured for 4 days. Alamar Blue readings were recorded every 24 h and relative cell viability of UBR5 deficient cells were compared to control cells on each day. b A549 cells were infected with shRNA against UBR5 and 1000 cells were cultured in 6-well plate for 10 days. Colonies were fixed in methanol and stained with crystal violet. c Quantitative evaluation of clonogenic assay. Representative bar graph showing number of colonies formed per 1000 cells

Article Snippet: Western blots were performed in Bolt Bis-Tris gels (#BG4120BOX, Life Technologies, Grand island, NY, USA) as per manufacturer’s protocol using antibodies from Santa Cruz, Dallas, TX, USA (GAPDH # sc47724); Bethyl, Montgomery, TX, USA (UBR5 # A300-573A, GCL1N1 # A301-843A) and Cell Signaling, Danvers, MA, USA (FLAG # 14793, DNA-PK # 4602, mTOR # 2972, RAPTOR # 2280, RICTOR # 2114, AKT # 4691, pAKT S473 # 4060).

Techniques: Infection, shRNA, Construct, Cell Culture, Control, Staining, Clonogenic Assay

Journal: BMC Cancer

Article Title: UBR-box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target

doi: 10.1186/s12885-020-07322-1

Figure Lengend Snippet: Loss of UBR5 results in reduction of tumor volume in NRGs mice. a A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5. Twenty-four hours post infection, cells were harvested and 1.25 × 10 5 cells were subcutaneously injected in mice flanks (L-NT shRNA & R-shRNA against UBR5). b Mice were euthanized 4 weeks after injection and tumor were measure. c Wilcoxon paired t-test were used to calculate the significance of difference between tumor volume

Article Snippet: Western blots were performed in Bolt Bis-Tris gels (#BG4120BOX, Life Technologies, Grand island, NY, USA) as per manufacturer’s protocol using antibodies from Santa Cruz, Dallas, TX, USA (GAPDH # sc47724); Bethyl, Montgomery, TX, USA (UBR5 # A300-573A, GCL1N1 # A301-843A) and Cell Signaling, Danvers, MA, USA (FLAG # 14793, DNA-PK # 4602, mTOR # 2972, RAPTOR # 2280, RICTOR # 2114, AKT # 4691, pAKT S473 # 4060).

Techniques: Infection, shRNA, Construct, Injection