Journal:

Article Title: The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

doi: 10.1128/JVI.76.24.12543-12552.2002

Figure Lengend Snippet: Representative cellular genes activated by BZLF1 expressiona

Article Snippet: The primary antibodies included anti-E2F-1 rabbit polyclonal antibody (diluted 1:500; C20 [Santa Cruz]), anti-cyclin E mouse monoclonal antibody (1:500; BF683 [Santa Cruz]), Cdc25A mouse monoclonal antibody (1:500; F-6 [Santa Cruz]), SLBP rabbit polyclonal antibody (1:2,000; a generous gift of William Marzluff, University of North Carolina at Chapel Hill), and anti-BZLF1 mouse monoclonal antibody (1:200; AZ-69 [Argene]).

Techniques: Activation Assay

Journal:

Article Title: The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

doi: 10.1128/JVI.76.24.12543-12552.2002

Figure Lengend Snippet: BZLF1 induces E2F-1 in a cell type-dependent manner. (A) Telomerase-immortalized human keratinocytes and normal human fibroblasts were infected with AdLacZ or AdBZLF1. At 48 h postinfection, the levels of E2F-1, BZLF1, and β-actin were quantitated by immunoblot analysis. (B) AGS gastric carcinoma cells and primary tonsil keratinocytes were infected as in panel A, and the level of E2F-1 was determined by immunoblot analysis. NS, nonspecific cross-reacting band. (C) FACS analysis was performed to compare the level of BZLF1 expression in Ad-BZLF1-infected AGS cells or telomerase-immortalized keratinocytes to that in the constitutively BZLF1-positive population of EBV-AGS cells. Neg., negative; Pos., positive.

Article Snippet: The primary antibodies included anti-E2F-1 rabbit polyclonal antibody (diluted 1:500; C20 [Santa Cruz]), anti-cyclin E mouse monoclonal antibody (1:500; BF683 [Santa Cruz]), Cdc25A mouse monoclonal antibody (1:500; F-6 [Santa Cruz]), SLBP rabbit polyclonal antibody (1:2,000; a generous gift of William Marzluff, University of North Carolina at Chapel Hill), and anti-BZLF1 mouse monoclonal antibody (1:200; AZ-69 [Argene]).

Techniques: Infection, Western Blot, Expressing

Journal:

Article Title: The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

doi: 10.1128/JVI.76.24.12543-12552.2002

Figure Lengend Snippet: BZLF1 increases the expression of E2F-1-responsive genes in some cell types. (A) Telomerase-immortalized keratinocytes or normal human fibroblasts were mock infected or infected with the AdLacZ or AdBZLF1 vector. Immunoblot analysis was performed 2 days later to quantitate expression of cyclin E, SLBP, or Cdc25A, as indicated. (B) AGS and primary tonsil keratinocytes were infected with the AdLacZ or AdBZLF1 vector, and immunoblot analysis was performed 2 days later to quantitate expression of cyclin E and SLBP as indicated.

Article Snippet: The primary antibodies included anti-E2F-1 rabbit polyclonal antibody (diluted 1:500; C20 [Santa Cruz]), anti-cyclin E mouse monoclonal antibody (1:500; BF683 [Santa Cruz]), Cdc25A mouse monoclonal antibody (1:500; F-6 [Santa Cruz]), SLBP rabbit polyclonal antibody (1:2,000; a generous gift of William Marzluff, University of North Carolina at Chapel Hill), and anti-BZLF1 mouse monoclonal antibody (1:200; AZ-69 [Argene]).

Techniques: Expressing, Infection, Plasmid Preparation, Western Blot

Journal:

Article Title: The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

doi: 10.1128/JVI.76.24.12543-12552.2002

Figure Lengend Snippet: BZLF1 induction of E2F-1 is not associated with apoptosis. Telomerase-immortalized human keratinocytes were mock infected or infected with AdLacZ or AdBZLF1. At 48 h postinfection, the binding of Annexin V-fluorescein isothiocyanate was quantitated by FACS analysis.

Article Snippet: The primary antibodies included anti-E2F-1 rabbit polyclonal antibody (diluted 1:500; C20 [Santa Cruz]), anti-cyclin E mouse monoclonal antibody (1:500; BF683 [Santa Cruz]), Cdc25A mouse monoclonal antibody (1:500; F-6 [Santa Cruz]), SLBP rabbit polyclonal antibody (1:2,000; a generous gift of William Marzluff, University of North Carolina at Chapel Hill), and anti-BZLF1 mouse monoclonal antibody (1:200; AZ-69 [Argene]).

Techniques: Infection, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Bcl3 deficiency enhances GSH recovery through Nrf2 signaling. ( A, B ) Bcl3 fl/fl and Bcl3 hep-/- mice were treated with APAP (300 mg/kg, intraperitoneally), and liver tissues were collected at 0, 3, 6, and 24 hours post-treatment. ( A ) Western blot analysis of Cyp2E1 levels in liver tissues. ( B ) Quantification of GSH levels in liver tissues using a GSH assay kit (n = 3–5 per group). ( C ) Immunohistochemical staining of GCLC in the livers of Bcl3 fl/fl and Bcl3 hep-/- mice treated with APAP (300 mg/kg, intraperitoneally) for 0 and 6 hours. ( D ) Western blot analysis of Nrf2, GCLC, and GCLM protein levels in liver tissues. ( E ) Quantification of protein levels. ( F ) qPCR analysis of Nrf2 , Gclc, and Gclm mRNA levels in liver tissues (n = 4–6 per group). Data represent 3 independent experiments and are presented as mean ± SEM. Statistical significance was determined by Student’s t-test ( B, E, F ); ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Western Blot, GSH Assay, Immunohistochemical staining, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Bcl3 deficiency mitigates APAP-induced primary hepatocyte death. ( A ) Primary hepatocytes were isolated from Bcl3 hep-/- mice and Bcl3 fl/fl mice. Representative images of primary hepatocytes treated with APAP (10 mM) for 0 and 6 hours. ( B ) Cell viability of primary hepatocytes measured using the CCK8 assay. ( C ) GSH levels in primary hepatocytes after APAP treatment for 3 and 6 hours. ( D ) MitoSOX staining in primary hepatocytes detected by immunofluorescence (scale bar, 10 μm). ( E ) Quantification of MitoSOX mean fluorescence intensity in primary hepatocytes by flow cytometry. ( F ) Protein levels of Cyp2E1, Nrf2, GCLC, and GCLM in primary hepatocytes treated with APAP for 3 and 6 hours, as determined by Western blotting. Data represent 3 independent experiments. Data are presented as mean ± SEM. Student’s t -test ( B, C ), ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Isolation, CCK-8 Assay, Staining, Immunofluorescence, Fluorescence, Flow Cytometry, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Bcl3 interacts with Nrf2. ( A ) Liver tissues from Bcl3 fl/fl and Bcl3 hep-/- mice treated with APAP (300 mg/kg intraperitoneally) for 0 and 6 hours were immunostained with anti-Bcl3 ( green ) and anti-Nrf2 ( red ) antibodies imaged using a confocal microscope (scale bar, 50 μm). ( B ) Primary hepatocytes isolated from Bcl3 hep-/- and Bcl3 fl/fl mice were immunostained for anti-Bcl3 ( green ) and anti-Nrf2 ( red ) following APAP treatment for 0 and 6 hours (scale bar, 10 μm). ( C–D ) Immunoprecipitation analysis of the interaction between Flag-Bcl3 and endogenous Nrf2 as well as Nrf2 and endogenous Bcl3 in Huh7 cells ( C ) and HEK293T cells ( D ) respectively. ( E ) Co-immunoprecipitation analysis using an anti-Bcl3 antibody to pull down Nrf2 protein in liver tissues from WT mice, followed by Western blotting.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Microscopy, Isolation, Immunoprecipitation, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Bcl3 knockout induces Nrf2 nuclear translocation following APAP treatment. ( A ) Liver tissues from Bcl3 fl/fl and Bcl3 hep-/- mice treated with APAP (300 mg/kg intraperitoneally) for 6 hours were immunostained for Nrf2 ( red ) (scale bar, 10 μm). ( B ) Primary hepatocytes isolated from Bcl3 hep-/- mice and Bcl3 fl/fl mice were immunostained for Nrf2 ( red ) following APAP treatment for 6 hours (scale bar, 10 μm). ( C ) Liver tissues from Bcl3 fl/fl and Bcl3 hep-/- mice treated with APAP (300 mg/kg intraperitoneally) for 6 hours were subjected to cytoplasmic and nuclear protein extraction. Nrf2 levels in cytoplasmic and nuclear fractions were analyzed by Western blotting (n = 3 per group). ( D ) Quantification of Western blot results. ( E ) Primary hepatocytes were isolated from the Bcl3 hep-/- mice and Bcl3 fl/fl mice. Nrf2 levels in cytoplasmic and nuclear fractions were analyzed by Western blotting (n = 3 per group). ( F ) Quantification of Western blot results. Data represent 3 independent experiments and are presented as mean ± SEM. Student t -test ( D, F ), ∗ P < .05; ∗∗ P < .01.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Knock-Out, Translocation Assay, Isolation, Protein Extraction, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: An Nrf2 inhibitor counteract the protective effect of Bcl3 on APAP-induced liver injury. ( A ) Experimental scheme. Bcl3 fl/fl and Bcl3 hep-/- mice were treated with the Nrf2 inhibitor ML385 (30 mg/kg/day intraperitoneally) for 5 days, fasted for 16 hours, and then administered APAP (300 mg/kg, intraperitoneally) for 24 hours before sacrifice and tissue collection. ( B ) Representative liver morphologies of mice from ( A ). ( C ) H&E staining of liver sections from mice in ( A ) (scale bar, 50 μm). ( D ) Quantification of necrotic areas in liver tissues (n = 5 per group). ( E ) Serum ALT levels (n = 5 per group). Data represent 3 independent experiments and are presented as mean ± SEM. Student t -test ( D, E ), ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Antibodies Used for Western Blot and Immunofluorescence

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Primers Used for qRT-PCR Analysis

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Sequencing

Journal: Nature communications

Article Title: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

doi: 10.1038/s41467-024-49431-6

Figure Lengend Snippet: Fig. 6 | UBE2D3 promotes KAP1 phosphorylation and telomere NHEJ in a PP2A- dependent manner. a Immunoblotting for pKAP1 (S824) in TRF2ts MEFs trans- duced with control, Ube2d3 and/or two independent Ppp2ca shRNAs at 32 °C or after 3 h at 37 °C. Representative blots from 2 independent experiments. b Quantification of chromosome fusions in TRF2ts MEFs transduced with control, Ube2d3 and two independent Ppp2ca shRNAs, upon 24 h of telomere uncapping. Two independent experiments are shown. c PP2A activity assays with immuno- precipitated PP2A from TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 3 h at 37 °C to induce telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Immunoblots of input and immunopre- cipitates are shown in Supplementary Fig. 8e. d PP2A phosphatase activity assay with immunoprecipitated PP2A-alpha (PP2Ac) from RNF168 mutant human cells (RIDDLE) with and without expression of ectopic HA-RNF168. Corrected for the amount of immunoprecipitated PP2A-alpha. Cells were untreated or harvested 30 min after irradiation with 3 Gy (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). The different symbols (dot, square and triangle)

Article Snippet: Primary antibodies used were against UBE2D3 (Y-25, sc-100618, SCBT, 1:500; 11677-1-AP, Proteintech, 1:500; 4330S, CST, 1:500 and A615, Boston Biochem, 1:2000), KAP1 (22553, Abcam, 1:1000), phospho-Kap1 S824 (A300 767A, Bethyl, 1:1000), 53BP1 (NB100-305, Novus, 1:500 and A300-272A, Bethyl, 1:2000), phospho-ATMS1981 (4526, CST, 1:1000), phospho-H2AX S139 (5636, Millipore, 1:1000), CHK2 (611570, BD, 1:500), c-myc (9E10, sc-40, SCBT, 1:250), HA (MMS101R, Covance, 1:1000), TRF2 (NB110-57130, Novus, 1:500), RNF8 (sc133971, SCBT, 1:250), MAD2L2 (14, sc-135977, SCBT, 1:500), GFP IgG fraction (A11122, Thermo Fisher Scientific, 1:1000), FLAG M2 (F1804, Sigma-Aldrich, 1:1000), Histone H3 (ab1791, Abcam, 1:10,000), hRNF168 (ABE367, Millipore, 1:500), hRNF168 (ABE467, Merck-Millipore, 1:1000), mRnf168 (gift from D. Durocher, 1:1000), hRIF1 (A300569A, Bethyl, 1:1000), mRIF1 (gift from S. Boulton and R. Chapman, 1:1000), Ligase 4 (H-300, sc-28232, SCBT, 1:300; NB110-57379, Novus, 1:500), FK2 (04-263, Millipore, 1:2000), HP1α (2616S, CST, 1:1000), Ubiquitin (P4D1, sc-8017, SCBT, 1:1000), HDAC1 (PA1-860, Thermo Fisher Scientific, 1:1000), PP2A C subunit, clone 1D6 antibody (05-421, Sigma-Aldrich/Millipore, 1:500), CDK4 (C-22, sc-260, SCBT, 1:500), HSP90 α/β (H-114, sc-7947, SCBT, 1:1000), γ-tubulin (T6557, SigmaAldrich, 1:10,000) β-actin (A5316, Sigma-Aldrich, 1:10,000), β-catenin (610154, BD, 1:10,000) and GAPDH (PA1-987, Thermo Fisher Scientific, 1:1000).HSP90α/β, γ-tubulin,β-actin,β-catenin andGAPDHwere used for loading controls.

Techniques: Phospho-proteomics, Western Blot, Control, Transduction, Activity Assay, Cell Culture, Two Tailed Test, Phosphatase Assay, Immunoprecipitation, Mutagenesis, Expressing, Irradiation

Journal: Antioxidants

Article Title: Selective Inhibition of Vascular Smooth Muscle Cell Function by COVID-19 Antiviral Drugs: Impact of Heme Oxygenase-1

doi: 10.3390/antiox14080945

Figure Lengend Snippet: RDV stimulates HO-1 promoter activity via the Nrf2/ARE complex in rat aortic SMCs. ( A ) RDV-mediated HO-1 mRNA expression is dependent on de novo RNA synthesis. SMCs were treated with actinomycin D (ActD; 1 µg/mL) for 8 h in the absence or presence of RDV (50 µM). ( B ) RDV stimulates HO-1 promoter activity. Cells were transfected with a HO-1 promoter construct (E1) or a mutated HO-1 construct (M739) and a Renilla luciferase construct, treated with RDV (50 µM) for 8 h, and then analyzed for luciferase activity. In some instances, a dominant-negative Nrf2 (dnNrf2) construct was co-transfected into cells. ( C ) RDV (50 µM), but not MPR (50 µM) or NTV (50 µM), exposure for 24 h increases ROS production in SMCs. ( D ) RDV (50 µM) exposure for 24 h stimulates ROS production that is blocked by the antioxidant N-acetyl-L-cysteine (NAC; 10 mM). ( E ) ChIP assays demonstrate that RDV (50 µM) exposure for 8 h increases Nrf2 binding to the HO-1 enhancer E1 that is blocked by NAC (10 mM). ( F ) NAC (10 mM) inhibits RDV (50 µM for 24 h)-mediated HO-1 protein expression. Statistical analysis was performed using an analysis of variance with the Holm–Sidak post hoc test. * Statistically significant effect of RDV.

Article Snippet: Polyclonal antibodies against HO-1 and HO-2 were from Enzo Life Sciences (Farmingdale, NY, USA) while antibodies directed against NF-E2-related factor-2 (Nrf2) and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Expressing, Transfection, Construct, Luciferase, Dominant Negative Mutation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: Analysis of the in vitro interaction between USP7-NTD and UbE2E1. A, schematics of UbE2E1 and USP7 indicating domain organization of the two proteins and the putative USP7 binding motif within the N terminus of UbE2E1. CAT, catalytic domain. B, comparison of the substrate peptide sequences recognized by USP7-NTD, featuring the (P/A)XXS motif, which is also found in the N terminus of UbE2E1. C, GST pulldowns were performed using GST-USP7-NTD and UbE2E1. GST-USP7-NTD fusion protein was incubated with UbE2E1, loaded onto glutathione resin (load (L)), washed with wash buffer (wash (W)), and eluted with Laemmli SDS-PAGE loading dye (eluate (E)). A GST pulldown of UbE2E1 protein with GST alone served as a negative control. GST pulldown experiments also tested the interaction of GST-USP7-NTD and the N-terminal UbE2E1 deletion mutant (ΔN-UbE2E1) and between the GST-USP7-NTD double mutant USP7-NTDDWand UbE2E1.

Article Snippet: Lysates were immunoprecipitated with an antibody against UbE2E1 (BD Biosciences) and immunoblotted with an antibody against USP7 (Bethyl Laboratories).

Techniques: In Vitro, Binding Assay, Incubation, SDS Page, Negative Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: Molecular analysis of the USP7-NTD-UbE2E1 interaction A, dissociation constants between USP7-NTD and UbE2E1 peptides were measured by intrinsic tryptophan fluorescence assays. B, the crystal structure of the USP7-NTD·UbE2E1ASTS complex. A ribbon representation of the structure of USP7-NTD (green) with UbE2E1ASTS in stick form (yellow) is shown. The 2Fo − Fc electron density map showing the UbE2E1ASTS peptide is contoured at 1σ. C, the molecular details of the interaction between USP7-NTD and UbE2E1ASTS are shown in stick format with the same color scheme as in B. Hydrogen bonds are indicated by black dashed lines. D, superimposition of UbE2E1ASTS (yellow) with the peptide from viral interferon regulatory factor protein 4, vIRF4ASTS (salmon), showing the similarity in mode of binding within the USP7 peptide binding region.

Article Snippet: Lysates were immunoprecipitated with an antibody against UbE2E1 (BD Biosciences) and immunoblotted with an antibody against USP7 (Bethyl Laboratories).

Techniques: Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 and UbE2E1 localization and interaction in vivo. A, immunofluorescence images of U2OS cells are shown after staining for endogenous UbE2E1 (Cy-3) and USP7 (Alexa Fluor 488) proteins. The merged image indicates a partially overlapping localization of USP7 and UbE2E1 in the nucleus. The nuclei were counterstained with DAPI. B, 293T cells transfected with FLAG-UbE2E1 were subject to immunoprecipitation (IP) using mouse IgG (negative control) and anti-FLAG followed by immunoblotting (IB) using antibodies against USP7 and UbE2E1. C, 293T cells transfected with Myc-USP7 were subject to immunoprecipitation using rabbit IgG and anti-Myc followed by immunoblotting against USP7 and UbE2E1 D, endogenous UbE2E1 from 293T cells was immunoprecipitated with a monoclonal antibody against UbE2E1 or mouse IgG followed by immunoblotting with polyclonal rabbit antibodies against USP7 or UbE2E1.

Article Snippet: Lysates were immunoprecipitated with an antibody against UbE2E1 (BD Biosciences) and immunoblotted with an antibody against USP7 (Bethyl Laboratories).

Techniques: In Vivo, Immunofluorescence, Staining, Transfection, Immunoprecipitation, Negative Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 attenuates UbE2E1-mediated ubiquitination. A, in vitro ubiquitination assays were performed using UbE2E1, UbE2D2, or ΔN-UbE2E1 (as the E2) in the presence of ATP, E1, Ub, the catalytic domain of NEDD4 (as the E3 ligase), and increasing amounts of USP7 (0–2 μg). Total ubiquitinated products (Ub(n))were detected by immunoblotting (IB) using a specific antibody against ubiquitin. Polyubiquitinated products were highlighted in boxes. B, ubiquitin loading assays were carried out in a reaction containing E1, His6-Ub, and ATP with increasing amounts of USP7 (0–2 μg) in the presence of 1 μg of full-length UbE2E1 or ΔN-UbE2E1. The UbE2E2-S∼Ub and ΔN-UbE2E2-S∼Ub intermediates were visualized by immunoblotting, using anti-UbE2E1 (left panel) or anti-His6 epitope tag (right panel). C, in vitro ubiquitination assays were performed as in A with the addition of active USP7 (lanes 3–5) or USP7 pretreated with ubiquitin-aldehyde (lanes 7–9), which blocks the catalytic activity of USP7. NC (negative control) represents a standard ubiquitination reaction in the absence of ATP.

Article Snippet: Lysates were immunoprecipitated with an antibody against UbE2E1 (BD Biosciences) and immunoblotted with an antibody against USP7 (Bethyl Laboratories).

Techniques: In Vitro, Western Blot, Activity Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 deubiquitinates UbE2E1. A, in vitro ubiquitination assays were performed as in Fig. 4A with increasing amounts of USP7. Total ubiquitination (Ub(n)) was detected using anti-Ub (left panel) and ubiquitinated UbE2E1 was detected using anti-UbE2E1 (right panel). NC (negative control) represents a standard ubiquitination reaction in the absence of ATP. IB, immunoblot. B, UbE2E1 is subject to ubiquitination and accumulated in the presence of MG132 in U2OS cells. Ubiquitinated UbE2E1 was immunoprecipitated (IP) and visualized by immunoblot using anti-ubiquitin. C, ubiquitinated UbE2E1 was subject to USP7 deubiquitination using USP7 or USP7-CS and visualized by immunoblot using anti-ubiquitin.

Article Snippet: Lysates were immunoprecipitated with an antibody against UbE2E1 (BD Biosciences) and immunoblotted with an antibody against USP7 (Bethyl Laboratories).

Techniques: In Vitro, Negative Control, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 stabilizes UbE2E1 in vivo. A, U2OS cells were transfected with nonspecific control siRNA (siControl) or siRNA targeting USP7 (siUSP7) and treated with 10 μg/ml cycloheximide (CHX) for the indicated number of hours or left untreated (lane 0). Cell lysates were subjected to SDS-PAGE and Western blot analysis using antibodies indicated. B, WT HCT116 cells or HCT116 USP7−/− were treated with cycloheximide and harvested for Western blotting as in A. The levels of UbE2E1 were normalized using actin as a loading control and presented as line graphs in the cycloheximide chase experiments. The asterisk represents the statistical analysis comparing levels of UbE2E1 after siRNA treatment with p < 0.01. Error bars indicate S.D. C, WT USP7 and the catalytically inactive mutant C223S, USP7-CS, were transfected into HCT116 USP7−/− cells. Cells were lysed and blotted for UbE2E1 48 h after transfection. The levels of UbE2E1 were normalized using actin as a loading control and presented in a bar graph (bottom). The asterisk represents the statistical analysis comparing levels of UbE2E1 after USP7 rescue with p < 0.01. Error bars indicate S.D.

Article Snippet: Lysates were immunoprecipitated with an antibody against UbE2E1 (BD Biosciences) and immunoblotted with an antibody against USP7 (Bethyl Laboratories).

Techniques: In Vivo, Transfection, SDS Page, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 regulates the stability of UbE2E1. USP7 attenuates UbE2E1-mediated total ubiquitination and stabilizes UbE2E1 through an interaction between USP7-NTD and the N-terminal extension of UbE2E1. Inactivation or disruption of the interaction between USP7 and UbE2E1 leads to UbE2E1 destabilization.

Article Snippet: Lysates were immunoprecipitated with an antibody against UbE2E1 (BD Biosciences) and immunoblotted with an antibody against USP7 (Bethyl Laboratories).

Techniques: