Journal: Physiological reports

Article Title: Inhibition of ubiquitin-specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts.

doi: 10.14814/phy2.14193

Figure Lengend Snippet: Figure 7. Effects of ML364 on cytotoxicity, proliferation, intracellular level of ATP, mitochondrial membrane potential, mitochondria morphology, and accumulation of ROS in C2C12 cells. C2C12 cells were treated with ML364 (10 µmol/L) or vehicle (DMSO) for 12 h (A), 5 days (B), 8 h (C, D), 4 h (E), or 0.5–2 h (F). (A) LDH content in the culture medium. (B) BrdU incorporation assay. Cells (500 cells) were seeded in a 96-well plate, incubated in the presence of BrdU with the inhibitor or vehicle for 5 days, and subjected to the BrdU ELISA assay. (C) Intracellular content of ATP. Values were calculated as ATP content per single cell, and are represented as relative to those detected in control cells. (D) The mitochondrial membrane potential was evaluated by staining with 20 nmol/L TMRM. The fluorescence intensity of cells was analyzed using a flow cytometer. After subtraction of the fluorescence intensity of nonstained cells, the median fluorescent signal values were calculated. (E) Morphology of mitochondria. After treatment with vehicle (left) or ML364 (right), the cells were stained with MitoTracker Red CMXRos. Scale bars represent 10 µm. (F) Mitochondrial accumulation of ROS. For the last 30 min of incubation, the cells were treated with the mitochondrial ROS indicator MitoSOX Red. Scale bars represent 20 µm. Nuclei were stained with Hoechst33342. Data are expressed as means one standard deviation of six (A-C) or seven (D) wells of a representative experiment of three experimental series.*P < 0.05 versus vehicle-treated cells (B-D). P-values obtained from Student’s t-test are also shown (A). Microscopic images are representative of three experiments (E, F).

Article Snippet: In some experiments, we treated the cells with the USP2-selective inhibitor ML364 (10 μmol/L) (MedChemExpress, Monmouth Junction, NJ, USA) or vehicle (DMSO, 2 mmol/L) for 0.5, 2, 4, 8, 12 h, or 5 days.

Techniques: Membrane, BrdU Incorporation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Single Cell, Control, Staining, Cytometry, Standard Deviation

Journal: Diabetes

Article Title: Deubiquitinating Enzyme USP2 Alleviates Muscle Atrophy by Stabilizing PPAR-γ

doi: 10.2337/db24-0375

Figure Lengend Snippet: Two lysine residues of PPAR-γ (184 and 185) are targeted for deubiquitination by USP2. A : The hot map of RNA-sequencing of Gas muscle of WT and USP2KO mice ( n = 5). B : Total cell lysates from the Gas muscle of mice subjected to Co-IP with anti-USP2 antibody and Western blots using indicated antibodies. C2C12 cells were transfected with Ad HA-Ppar-γ and/or Ad-Flag Usp2 WT, as indicated. Total cells lysates were subjected to Co-IP with anti-Flag antibody; Western blotting results used the indicated antibodies. C : Human skeletal muscle cells (HSkMCs) transfected with Ad-Flag USP2 WT (Flag-WT) or Ad-Flag USP2 C276A. Total cell lysates were subjected to Co-IP with anti-PPAR-γ antibody; Western blots using indicated antibodies, and the charts report the quantitative result. D : WT or USP2 knockdown HSkMCs transfected with Ad-Flag PPAR-γ WT, K184R, K185R, K268R, K293R, or K462R as indicated. The total cell lysates were prepared; Western blotting used the indicated antibodies; chart reports the quantitative result. * P < 0.05, ** P < 0.01, *** P < 0.001, by unpaired Student t test. E : WT or USP2 knockdown HSkMCs transfected with Ad-Flag PPAR-γ WT and DKR (both K184R and K185R) as indicated. Total cell lysates subjected to Co-IP with anti-Flag antibody; Western blots using indicated antibodies and charts of the quantitative results are shown. F : USP2–PPAR-γ docking with the HDOCK server. High magnification of boxed areas is presented on the right in each row. Arrow indicates PPAR-γ protein K184 and K185 site. Data are expressed as mean ± SD. B , C , and E : * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA with Bonferroni correction.

Article Snippet: Usp2 flox/flox mice (C57BL/6JCya- Usp2 em1flox/Cya; strain no. S-CKO-11600) were purchased from Cyagen Biosciences (Guangzhou, Guangdong, China).

Techniques: RNA Sequencing, Co-Immunoprecipitation Assay, Western Blot, Transfection, Knockdown

Journal: Diabetes

Article Title: Deubiquitinating Enzyme USP2 Alleviates Muscle Atrophy by Stabilizing PPAR-γ

doi: 10.2337/db24-0375

Figure Lengend Snippet: USP2 improves insulin resistance in skeletal muscle. A – C : Immunoblot analysis of PPAR-γ, GLUT4, IRS1, and tubulin in Gas from mice as indicated. The chart presents the levels of the indicated protein normalized to tubulin ( n = 6). D : Immunoblot analysis of PPAR-γ, USP2, GLUT4, IRS1, and tubulin in C2C12 myotubes transfected with Ctrl or Usp2 siRNA (si Usp2 ) in the presence or absence of TNF-α at 20 ng/mL for 24 h. The chart in the middle presents the levels of the indicated protein normalized to tubulin ( n = 3). The chart at the right reports the qPCR analysis of Atrogin1, MUSA1 , and F-box protein 31 ( Fbxo31 ) in C2C12 myotubes transfected with Ctrl or Usp2 siRNA and treated with or without TNF-α at 20 ng/mL for 24 h ( n = 5). E : MYHC immunofluorescence of C2C12 myotubes transfected with Ctrl or Usp2 siRNA and treated with or without TNF-α at 20 ng/mL for 24 h. The chart presents the levels of fusion index and myotube diameter ( n = 5). F : Immunoblot analysis of PPAR-γ, USP2, GLUT4, IRS1, and tubulin in C2C12 myotubes infected with adenovirus expressing Usp2 (Ad Usp2 ) or green fluorescent protein (Ctrl). Myotubes were cultured for 24 h in the presence or absence of TNF-α at 20 ng/mL. The middle chart presents the levels of the indicated protein normalized to tubulin ( n = 3). The chart on the right presents results of the qPCR analysis of Atrogin1, MUSA1 , and Fbxo31 in C2C12 myotubes infected with Ad Usp2 or Ctrl and treated with or without TNF-α at 20 ng/mL for 24 h ( n = 5). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA with Bonferroni correction. Vehi, vehicle.

Article Snippet: Usp2 flox/flox mice (C57BL/6JCya- Usp2 em1flox/Cya; strain no. S-CKO-11600) were purchased from Cyagen Biosciences (Guangzhou, Guangdong, China).

Techniques: Western Blot, Transfection, Immunofluorescence, Infection, Expressing, Cell Culture

Journal: Diabetes

Article Title: Deubiquitinating Enzyme USP2 Alleviates Muscle Atrophy by Stabilizing PPAR-γ

doi: 10.2337/db24-0375

Figure Lengend Snippet: USP2-regulated insulin signaling depends on PPAR-γ in vitro. A : Immunoblot analysis of USP2, PPAR-γ, IRS1, GLUT4, and tubulin in C2C12 myotubes transfected with Ctrl or Pparγ siRNA (si Pparγ ) in the presence or absence of TNF-α at 20 ng/mL for 24 h. Charts present the levels of the indicated protein normalized to tubulin ( n = 3). B : qPCR analysis of Ccng2 , Cdkn1b , Rbl2 , Bnip3, Atrogin1, MUSA1 , and F-box protein 31 ( Fbxo31 ) in C2C12 myotubes ( n = 5). C : MYHC immunofluorescence of C2C12 myotubes transfected with si Usp2 and/or si Pparγ in the presence or absence of TNF-α at 20 ng/mL for 24 h. Charts present the levels of fusion index ( n = 5). D : Immunoblots analysis of USP2, PPAR-γ, IRS1, GLUT4, and tubulin in C2C12 myotubes infected with adenovirus expressing Usp2 (Ad Usp2 ) and/or si Pparγ as indicated. Myotubes were cultured for 24 h in the presence or absence of DEX at 50 μmol/L. Charts present the levels of the indicated protein normalized to tubulin ( n = 3). E : qPCR analysis of Ccng2 , Cdkn1b , Rbl2 , Bnip3, Atrogin1, MUSA1 , and Fbxo31 in C2C12 myotubes infected with Ad Usp2 and/or si Pparγ and treated with or without DEX at 50 μmol/L for 24 h ( n = 5). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA with Bonferroni correction.

Article Snippet: Usp2 flox/flox mice (C57BL/6JCya- Usp2 em1flox/Cya; strain no. S-CKO-11600) were purchased from Cyagen Biosciences (Guangzhou, Guangdong, China).

Techniques: In Vitro, Western Blot, Transfection, Immunofluorescence, Infection, Expressing, Cell Culture

Journal: Diabetes

Article Title: Deubiquitinating Enzyme USP2 Alleviates Muscle Atrophy by Stabilizing PPAR-γ

doi: 10.2337/db24-0375

Figure Lengend Snippet: PPAR-γ-inhibition abolished the effects of USP2 KO on aggravating insulin resistant and alleviating muscle fiber atrophy. We established a DM-induced muscle atrophy model in WT mice transfected with AAV-sh Pparγ and/or AAV- Usp2 , as indicated. n = 6. A : Western blot analysis of USP2, PPARγ, MURF-1, MYHC, and tubulin in the Gas muscle of mice. Charts present the quantification results. B : The FBG and FBI of mice. C : The ratio of Gas muscle and TA muscle weight to body weight. D : Grip strength test and exhaustive running distance results. E : Representative images of myofiber cross-sections obtained through hematoxylin-eosin (H-E) staining (scale bar = 100 μmol/L) (left) and cross-sectional area (CSA) of Gas muscles from mice (right). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA with Bonferroni correction. Vehi, vehicle.

Article Snippet: Usp2 flox/flox mice (C57BL/6JCya- Usp2 em1flox/Cya; strain no. S-CKO-11600) were purchased from Cyagen Biosciences (Guangzhou, Guangdong, China).

Techniques: Inhibition, Transfection, Western Blot, Staining, Muscles

Journal: Nature Chemical Biology

Article Title: Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36

doi: 10.1038/s41589-023-01388-1

Figure Lengend Snippet: a . Schematic representation of human USP36 domain architecture with active site residues shown as stars. The protein boundaries used for crystallography studies are indicated. b . Schematic representation of human USP16 domain architecture. The protein boundaries used for in-vitro studies are indicated. The USP domain of USP16 contains various large insertions . c.-f . Quantification of Ubiquitin and Fubi C-terminal hydrolase activity using Ub-RhoG and Fubi-RhoG substrates, respectively, for both USP2 ( c , d ) and USP30 ( e , f ) as described in Fig. .

Article Snippet: Human Fubi 1–73 , HA–Fubi 1–73 , GST–3C-GS-HA–Fubi 1–73 , ubiquitin 1–75 , HA–ubiquitin 1–75 , HA–NEDD8 1-75 , HA–ISG15 1–156 , HA–UFM1 1–82 , HA–URM1 1–100 and HA–SUMO1 1–96 were cloned from Escherichia coli optimized DNA gene strings into pTXB1 using restriction enzymes. cDNA encoding catalytic domains of human USP16 191–823 (Medical Research Council Protein Phosphorylation and Ubiquitylation (MRC PPU), DU25374), USP42 70–446 (MRC PPU, DU15140), USP36 81–461 and USP36 81–424 (MRC PPU, DU49030) were cloned into pOPINK, and USP2 258–605 (Addgene, 22577), USP8 734–1110 (Addgene, 22608) and USP18 16–372 (MRC PPU, DU14320) were cloned into pOPINB using an In-Fusion HD cloning kit (Takara Clonetech).

Techniques: In Vitro, Ubiquitin Proteomics, Activity Assay

Journal: Nature Chemical Biology

Article Title: Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36

doi: 10.1038/s41589-023-01388-1

Figure Lengend Snippet: a , Semisynthesis of the Fubi-PA probe from Fubi-MesNa (top) and characterization of these species by intact protein MS (bottom). b , Reactivity assessment of recombinant USP16 incubated with the indicated probes for 1 h at 37 °C. Chloroacetamide (CAA) pretreatment was performed where indicated. Protein samples were analyzed by SDS–PAGE and Coomassie staining; ABP, activity-based probe. c , Reactivity assessment of recombinant USP36 as described in b . d , Results of a DUB panel inhibition assay shown as a heat map. Recombinant DUBs were incubated with Fubi-PA (4 µM) for 15 min at room temperature, and their activities were subsequently assessed. Residual activities for USP16 and USP36 are given as numbers. Data show results from technical duplicates normalized for each DUB to its respective activity in the absence of probe; UCHs, ubiquitin carboxy-terminal hydrolases; OTUs, ovarian tumor family of DUBs. e , Reactivity assessment of recombinant USP2 as described in b and c . f – h , Thermal stability assessment of USP36 ( f ), USP16 ( g ) and USP2 ( h ) complexed with Fubi or ubiquitin probes. Means and individual results of three samples are plotted; T m , protein melting temperature.

Article Snippet: Human Fubi 1–73 , HA–Fubi 1–73 , GST–3C-GS-HA–Fubi 1–73 , ubiquitin 1–75 , HA–ubiquitin 1–75 , HA–NEDD8 1-75 , HA–ISG15 1–156 , HA–UFM1 1–82 , HA–URM1 1–100 and HA–SUMO1 1–96 were cloned from Escherichia coli optimized DNA gene strings into pTXB1 using restriction enzymes. cDNA encoding catalytic domains of human USP16 191–823 (Medical Research Council Protein Phosphorylation and Ubiquitylation (MRC PPU), DU25374), USP42 70–446 (MRC PPU, DU15140), USP36 81–461 and USP36 81–424 (MRC PPU, DU49030) were cloned into pOPINK, and USP2 258–605 (Addgene, 22577), USP8 734–1110 (Addgene, 22608) and USP18 16–372 (MRC PPU, DU14320) were cloned into pOPINB using an In-Fusion HD cloning kit (Takara Clonetech).

Techniques: Recombinant, Incubation, SDS Page, Staining, Activity Assay, Inhibition, Ubiquitin Proteomics

Journal: Nature Chemical Biology

Article Title: Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36

doi: 10.1038/s41589-023-01388-1

Figure Lengend Snippet: a , Cartoon and transparent surface representation of Fubi in two orientations colored according to sequence conservation, which was calculated from sequences of 156 species. Selected residues are shown as sticks and are labeled. b – f , Reactivity assessment of recombinant proteins USP36 ( b ), USP16 ( c ), USP2 ( d ), USP42 ( e ) and USP30 ( f ) with wild-type ubiquitin-PA, wild-type Fubi-PA or Fubi-PA probes carrying the indicated mutations (which substitute the ubiquitin-equivalent residues into Fubi). Cross-reactivity to mutated Fubi probes is shown with black arrowheads for USP42 and USP30. Data are representative of three independent experiments. g , Model summarizing important motifs on Fubi underlying the Fubi/ubiquitin cross-reactivity in USP16/USP36 and the restriction from other DUBs, thereby enabling spatially controlled maturation of Fubi-S30. h , Schematic representation of the proposed two-tier processing of Fubi-S30 and the Fubi system.

Article Snippet: Human Fubi 1–73 , HA–Fubi 1–73 , GST–3C-GS-HA–Fubi 1–73 , ubiquitin 1–75 , HA–ubiquitin 1–75 , HA–NEDD8 1-75 , HA–ISG15 1–156 , HA–UFM1 1–82 , HA–URM1 1–100 and HA–SUMO1 1–96 were cloned from Escherichia coli optimized DNA gene strings into pTXB1 using restriction enzymes. cDNA encoding catalytic domains of human USP16 191–823 (Medical Research Council Protein Phosphorylation and Ubiquitylation (MRC PPU), DU25374), USP42 70–446 (MRC PPU, DU15140), USP36 81–461 and USP36 81–424 (MRC PPU, DU49030) were cloned into pOPINK, and USP2 258–605 (Addgene, 22577), USP8 734–1110 (Addgene, 22608) and USP18 16–372 (MRC PPU, DU14320) were cloned into pOPINB using an In-Fusion HD cloning kit (Takara Clonetech).

Techniques: Sequencing, Labeling, Recombinant, Ubiquitin Proteomics

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability

doi: 10.1096/fj.202001146RR

Figure Lengend Snippet: Inhibition of neddylation stabilizes antithrombin. A,B, Assessment of antithrombin expression in HepG2 and SMMC7721 cells after exposure to the indicated concentrations of MLN4924 for 24 hours or to 1 µM MLN4924 for the indicated times. A, Representative western blots showing antithrombin protein expression. B, Statistical analysis of antithrombin protein expression. C, The half-life of antithrombin was evaluated by exposing HepG2 and SMMC7721 cells to CHX alone or in combination with MLN4924 for the indicated times. D, HepG2 and SMMC7721 cells were treated with MLN4924 for 24 hours, followed by treatment with 100 nM bortezomib for an additional 2 hours. Total polyubiquitinated antithrombin was evaluated by immunoprecipitation. E-G, Western blot analysis of the neddylation proteins, UBA3 (E), NAE1 (F), and UBE2M (G), in HepG2 and SMMC7721 cells 48 hours after transfection with 50 nM siRNAs against the corresponding enzymes

Article Snippet: Medium was replaced with fresh medium, as appropriate, after transfection for 6 hours and cells were cultured for 48 or 72 hours for further analysis. siRNAs against USP2 (sc-76821), UBA3 (sc-76783), NAE1 (sc-72523), and UBE2M (sc-76786) were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Expressing, Western Blot, Immunoprecipitation, Transfection

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability

doi: 10.1096/fj.202001146RR

Figure Lengend Snippet: Cullin 2 regulates the ubiquitination and degradation of antithrombin. A,B, Endogenous antithrombin was immunoprecipitated and immunoblotted for the indicated proteins in HepG2 cells. Cullin 2 (A) and RBX1 (B) were detected in immunoprecipitates. C, Evaluation of the interaction of endogenous Cullin 2 with antithrombin in HepG2 and SMMC7721 cells by immunoprecipitation. D, Western blot analysis of Cullin 2 in HepG2 and SMMC7721 cells 72 hours after transfection with 100 nM siRNAs against Cullin 2. E, Western blot analysis of Cullin 2 in HepG2 and SMMC7721 cells transfected with 100 nM siRNAs against Cullin 2 as in D, followed by treatment with 100 nM bortezomib for an additional 2 hours. Total polyubiquitinated antithrombin was assessed by immunoprecipitation

Article Snippet: Medium was replaced with fresh medium, as appropriate, after transfection for 6 hours and cells were cultured for 48 or 72 hours for further analysis. siRNAs against USP2 (sc-76821), UBA3 (sc-76783), NAE1 (sc-72523), and UBE2M (sc-76786) were purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability

doi: 10.1096/fj.202001146RR

Figure Lengend Snippet: USP2 regulates antithrombin ubiquitination and degradation. A, Immunoprecipitation followed by western blotting showing the association of antithrombin with endogenous USP2, but not USP30, in HepG2 cells. B, Interaction of endogenous USP2 and antithrombin in HepG2 and SMMC7721 cells, assessed by immunoprecipitation. C, Western blotting of antithrombin and USP2 proteins in 293FT cells transfected with FLAG-antithrombin constructs following siRNA (100 nM)-mediated knockdown of USP2. D, Western blotting of endogenous USP2 and antithrombin in HepG2 and SMMC7721 cells transfected with siRNA against USP2 (or control siRNA). E, RT-qPCR analysis of endogenous (HepG2 and SMMC7721 cells) and exogenous (293FT cells) antithrombin and endogenous USP2 mRNA levels. F, Pulse-chase analysis of antithrombin expression in HepG2 and SMMC7721 cells transfected with siRNA (100 nM) against USP2 (or control siRNA) then treated with CHX (50 µg/mL) for the indicated times. G, 293FT cells were transfected with FLAG-antithrombin and siRNA against USP2 (or control siRNA) followed by treatment with bortezomib (10 µM) for 6 hours. Total and K48-linked polyubiquitinated antithrombin was detected by immunoprecipitation using an anti-FLAG antibody followed by western blotting

Article Snippet: Medium was replaced with fresh medium, as appropriate, after transfection for 6 hours and cells were cultured for 48 or 72 hours for further analysis. siRNAs against USP2 (sc-76821), UBA3 (sc-76783), NAE1 (sc-72523), and UBE2M (sc-76786) were purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection, Construct, Knockdown, Control, Quantitative RT-PCR, Pulse Chase, Expressing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability

doi: 10.1096/fj.202001146RR

Figure Lengend Snippet: USP2 overexpression stabilizes antithrombin. A, Western blotting of antithrombin and USP2 expression in 293FT cells transfected with GFP-antithrombin, with or without bortezomib (1 μM) treatment for 6 hours, or co-transfected with HA-USP2 WT or catalytically inactive (C276A) HA-USP2. B, Western blot analysis of antithrombin and USP2 expression in 293FT cells transfected with GFP-antithrombin and increasing concentrations of HA-USP2. C, Western blot analysis of antithrombin and USP2 expression in HepG2 and SMMC7721 cells overexpressing HA-USP2 (or control vector). D, Pulse-chase analysis of antithrombin expression in HepG2 and SMMC7721 cells overexpressing HA-USP2 (or control vector) and treated with CHX (50 μg/mL) for additional indicated times. E, Total and K48-linked polyubiquitinated antithrombin in 293FT cells transfected with GFP-antithrombin and HA-USP2 WT or HA-USP2C276A and treated 48 hours later with bortezomib (10 μM) for 6 hours, detected by immunoprecipitation and subsequent western blotting. F, Total and K48-linked polyubiquitinated antithrombin in HepG2 cells overexpressing HA-USP2 WT or HA-USP2C276A, treated for 6 hours with bortezomib (10 μM) 48 hours after transfection, followed by immunoprecipitation and subsequent western blotting

Article Snippet: Medium was replaced with fresh medium, as appropriate, after transfection for 6 hours and cells were cultured for 48 or 72 hours for further analysis. siRNAs against USP2 (sc-76821), UBA3 (sc-76783), NAE1 (sc-72523), and UBE2M (sc-76786) were purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Pulse Chase, Immunoprecipitation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability

doi: 10.1096/fj.202001146RR

Figure Lengend Snippet: Scheme showing that Cullin 2 ubiquitin ligase and USP2 coordinate antithrombin ubiquitination and degradation

Article Snippet: Medium was replaced with fresh medium, as appropriate, after transfection for 6 hours and cells were cultured for 48 or 72 hours for further analysis. siRNAs against USP2 (sc-76821), UBA3 (sc-76783), NAE1 (sc-72523), and UBE2M (sc-76786) were purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Targeting USP2 regulation of VPRBP-mediated degradation of p53 and PD-L1 for cancer therapy

doi: 10.1038/s41467-023-37617-3

Figure Lengend Snippet: a Western blot analysis for VPRBP after immunoprecipitation (IP) of FH-USP2, with HA beads, from H1299 cells transfected with Myc-VPRBP alone or with FH-USP2. b Western blot analysis for USP2 after immunoprecipitation (IP) of SFB-VPRBP, with streptavidin beads, from H1299 cells transfected with FH-USP2 alone or with SFB-VPRBP. c Western blot analysis for endogenous VPRBP after immunoprecipitation of endogenous USP2 and control IgG in A375 cells. d Western blot analysis of F-VPRBP pulled down by GST or GST-USP2 protein in in vitro GST-pull down assay. e Western blot analysis of Myc-VPRBP in H1299 whole cell extracts transfected with Myc-VPRBP alone, or plus Myc-USP2 wild-type (WT) or C276A. f Western blot analysis of ubiquitinated VPRBP (Ub(n)-VPRBP) after immunoprecipitation of SFB-VPRBP by S protein beads under denaturing condition in H1299 cells transfected with indicated constructs. g , h Cal33 and H1299 cells were transfected with control or USP2 siRNA for 96 h. Whole cell lysates were subjected to SDS-PAGE followed by western blot analysis ( g ). Total RNA was extracted for cDNA synthesis and qPCR analysis ( h ). n = 3 biologically independent samples, mean ± SD, two-tailed unpaired t -test. i Western blot analysis of VPRBP in H1299 cells transfected with control or USP2 siRNA oligos. j Western blot analysis of VPRBP and MDM2 in H1299 control or USP2 knockout (KO) cells. k , l H1299 control and USP2 knockout (KO) cells were treated with 100 μg/ml cycloheximide (CHX) for indicated time. Whole cell extracts were subjected to SDS-PAGE followed by western blot analysis ( k ). VPRBP protein abundance was quantified with Image J software ( l ). All data are representative of at least two independent experiments. Source data are provided in the Source data file.

Article Snippet: H1299 control and USP2 knockout cells were generated by using similar method with control double nickase plasmid (Santacruz biotechnology, sc-437281) and USP2 double nickase plasmid (Santacruz biotechnology, sc-411243-NIC).

Techniques: Western Blot, Immunoprecipitation, Transfection, Control, In Vitro, Pull Down Assay, Construct, SDS Page, cDNA Synthesis, Two Tailed Test, Knock-Out, Quantitative Proteomics, Software

Journal: Nature Communications

Article Title: Targeting USP2 regulation of VPRBP-mediated degradation of p53 and PD-L1 for cancer therapy

doi: 10.1038/s41467-023-37617-3

Figure Lengend Snippet: a , b H460, A375 ( a ), and H1299 ( b ) cells were treated with 20 μM ML364 for 72 h. Whole cell extracts were subjected to SDS-PAGE followed by western blot analysis. c U2OS and H1299 cells were treated with 20 μM LCAHA for 72 h. Whole cell extracts were subjected to SDS-PAGE followed by western blot analysis. d H1299 cells were pretreated with DMSO, 1 μM, 2.5 μM, and 10 μM RG7388 or ML364 for 30 min and then transfected with F-P53 alone, or plus F-MDM2 for 24 h. Whole cell extracts were subjected to SDS-PAGE followed by western blot analysis. e H1299 control and USP2 knockout (KO) cells were treated with 100μg/ml cycloheximide (CHX) for indicated time. Whole cell extracts were subjected to SDS-PAGE followed by western blot analysis. f Western blot analysis in H1299 control or USP2 knockout (KO) cells transfected with F-P53 alone or plus increasing amounts of F-MDM2 constructs. g Western blot analysis of endogenous P21 and MDM2 expression in H1299 control or USP2 knockout (KO) cells transfected with or without F-P53 constructs. All data are representative of two independent experiments. Source data are provided in the Source data file.

Article Snippet: H1299 control and USP2 knockout cells were generated by using similar method with control double nickase plasmid (Santacruz biotechnology, sc-437281) and USP2 double nickase plasmid (Santacruz biotechnology, sc-411243-NIC).

Techniques: SDS Page, Western Blot, Transfection, Control, Knock-Out, Construct, Expressing

Journal: iScience

Article Title: Cellular targets and lysine selectivity of the HERC5 ISG15 ligase

doi: 10.1016/j.isci.2024.108820

Figure Lengend Snippet: Di-G proteomics of IFN-β-treated A549 cells (A) A549 or A549- HERC5 KO cells were treated with IFN-β as indicated and total cell extracts were immunoblotted with antibodies specific for HERC5, ISG15, and β-actin. (B) Schematic overview of the Di-G proteomics workflow for characterizing the ISGylome of IFN-β-treated A549 cells. (C) Cell extracts from IFN-β-treated A549 and A549- HERC5 KO cells were immunoblotted for ubiquitin (left) or ISG15 (right), before and after treatment with USP2-cc. (D) Unsupervised clustering of 4,001 di-G peptides identified in extracts from IFN-β-treated A549- HERC5 KO and A549 cells (three replicates each).

Article Snippet: pET28a-LIC USP2 , Addgene , CAT# 36894); RRID:Addgene_36894.

Techniques: Ubiquitin Proteomics

Journal: iScience

Article Title: Cellular targets and lysine selectivity of the HERC5 ISG15 ligase

doi: 10.1016/j.isci.2024.108820

Figure Lengend Snippet: Identification of targets of hHERC5 and mHERC6 by expression of conjugation components in HEK293T-HERC5 KO cells (A) Top: HEK293T- HERC5 KO cells were transfected with plasmids expressing FLAG-hISG15, hUBA7, hUBE2L6, with or without hHERC5. Cell extracts were probed by immunoblotting for ubiquitin (left) or FLAG-ISG15 (right), before and after USP2-cc treatment. Bottom: as above, except transfected plasmids were FLAG-mISG15, hUBA7, mUBE2L6, with or without mHERC6. (B) Unsupervised hierarchical clustering of 16,035 modified lysines identified in cells expressing the human ISGylation enzymes with and without hHERC5 and the mouse ISGylation enzymes with and without mHERC6 (three replicates of each transfection condition). Red indicates identified sites and gray indicates that the site was not identified. Five major clusters (C1-C5) are indicated; see text for complete description. (C) Venn diagrams showing overlap of proteins that are ISGylated by hHERC5 and mHERC6 targets (left) and overlap of hHERC5 and mHERC6 ISGylation sites (right). (D) Uniform manifold approximation and projection (UMAP) were used to visualize the lysine preferences of hHERC5 in A549 IFN-β-induced cells (purple) and in HEK293T-transfected cells (red), as well as the lysine preferences of mHERC6 (pink) and total ubiquitylation site preferences in the HEK293T datasets (blue). Each point corresponds to one of the three experimental replicates for each condition.

Article Snippet: pET28a-LIC USP2 , Addgene , CAT# 36894); RRID:Addgene_36894.

Techniques: Expressing, Conjugation Assay, Transfection, Western Blot, Ubiquitin Proteomics, Modification

Journal: iScience

Article Title: Cellular targets and lysine selectivity of the HERC5 ISG15 ligase

doi: 10.1016/j.isci.2024.108820

Figure Lengend Snippet:

Article Snippet: pET28a-LIC USP2 , Addgene , CAT# 36894); RRID:Addgene_36894.

Techniques: Recombinant, Transfection, CRISPR, Ubiquitin Proteomics, Peptide Fractionation, Mass Spectrometry, Expressing, Plasmid Preparation, Software

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: The association between USP2 expression and clinicopathological parameters in bladder cancer patients.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Abnormally elevated expression of USP2 in BLCA tissues and cell lines. (A) RT-qPCR analysis of USP2 level in tumor tissues and adjacent normal tissues of 46 BLCA patients. (B) The protein level of USP2 was examined in 4 BLCA patients. (C) RT-qPCR was used to test USP2 expression in BLCA tissues. ΔCTN represented comparative Ct in normal tissues. ΔCTC was comparative Ct in tumor tissues. (D) Kaplan-Meier survival analysis of the relationship between USP2 level and overall survival of BLCA patients. (E, F) RT-qPCR and western blot analysis of USP2 level in BLCA cell lines and normal bladder epithelial cells SV-HUC-1. Values are mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Depletion of USP2 weakened cell proliferation, migration and invasion of BLCA cells. (A, B) USP2 expression was extremely decreased in 5637 and J82 cells transfected with sh-USP2#1 or sh-USP2#2. (C) CCK-8 assay displayed cell growth curves in 5637 and J82 cells. (D) Representative images and quantification of EdU assay were performed in BLCA cells. (E) The migration of BLCA cells was assessed using the wound healing assay. (F) Transwell assay was used to test the capacity of cell invasion in BLCA cells. Results expressed as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Migration, Expressing, Transfection, CCK-8 Assay, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: EZH2 was increased in BLCA and correlated with USP2 level. (A) RT-qPCR analysis of EZH2 level in tumor tissues and adjacent normal tissues of 46 BLCA patients. (B) RT-qPCR was used to test EZH2 expression in BLCA tissues. (C) Kaplan-Meier survival analysis of the relationship between EZH2 level and overall survival of BLCA patients. (D) The correlation between USP2 and EZH2 levels in BLCA tissues was assessed using Spearman analysis. (E) RT-qPCR analysis of EZH2 level in BLCA cell lines and normal bladder epithelial cells. (F) RT-qPCR detection of EZH2 expression in 5637 and J82 cells transfected with sh-USP2. (G) Western blot analysis of EZH2 and H3K27me3 levels in 5637 and J82 cells transfected with sh-USP2. Data are represented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: USP2 stabilized EZH2 by inhibiting its ubiquitination and degradation in BLCA cells. (A) 5637 and J82 cell lysates were immunoprecipitated using antibodies against USP2 or EZH2, followed by immunoblotting with the indicated antibodies. (B) BLCA cells were transfected with His-USP2 and HA-EZH2. Cell lysates were immunoprecipitated using anti-His magnetic beads, and immunoblotted with His and HA antibodies. (C) Cells overexpressing USP2 were treated with 50 ng/ml Cycloheximide (CHX) for 3, 6, and 9 h. Western blot was used to detect EZH2 expression. (D) Western blot was used to detect the expressions of USP2 and EZH2 in cells transfected with sh-NC or sh-USP2 with/without proteasomal inhibitor MG132 (20 μM) for 5 h. (E) EZH2 in BLCA cells transfected with His-USP2 or sh-USP2 was immunoprecipitated to detect ubiquitylation. Data are represented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection, Magnetic Beads, Expressing

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Overexpressed EZH2 abolished USP2 silencing-mediated biological functions on BLCA cells. We knocked down the level of USP2 and overexpressed EZH2 expression in 5637 cells. (A) The CCK-8 assay displayed cell growth curves in 5637 cells. (B) Representative images and quantification of EdU assay in 5637 cells. (C) Transwell detection of invasion in 5637 cells. (D) Western blot analysis of USP2 and EZH2 protein levels. Values were expressed as mean ± SD of three separate determinations. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, CCK-8 Assay, EdU Assay, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: USP2 epigenetically repressed SOX1 expression via EZH2 stabilization. RT-qPCR was used to detect the levels of candidate gene in BLCA cells transfected with sh-USP2 (A) or si-EZH2 (B). (C, D) ChIP assay was performed to assess the occupancy of EZH2 and H3K27me3 in the SOX1 promoter region in cells with sh-NC or sh-USP2. (E, F) RT-qPCR and western blot analysis of EZH2 and SOX1 expression in BLCA cells transfected with sh-USP2 and/or pcDNA3.1-EZH2. Error bars stand for the mean ± SD of at least triplicate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: The USP2-EZH2-SOX1 axis in tumor growth of BLCA in vivo . The 5637 cells stably expressing sh-USP2 or EZH2 were separately injected into the flanks of BALB/c nude mice to establish a xenograft tumor model. (A) Representative photographs of tumors from the xenograft nude mice. (B) Tumor weight in the three groups. (C) Tumor volumes were determined on the indicated days. (D) IHC analysis detected the expression of SOX1 in tumor tissues. (E) Schematic graph illustrating the role of USP2 in regulating the progression of BLCA. The results are representative of three independent experiments. All data are represented as mean ± SD. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: In Vivo, Stable Transfection, Expressing, Injection

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2 protein expression levels were reduced in HCC-T tissues from HCC patients. (A) Representative total USP2 protein staining with goat anti-human USP2 IgG in the tissues of human normal liver (n=76), HCC-NT (n=38) and HCC-T (n=115) by IHC. As negative controls, human normal liver tissues (n=6) were also stained with goat pre-immune serum. (B) Quantification of the USP2 protein levels were performed with ImageJ. (C) Representative USP2 protein staining in four paired HCC-T and HCC-NT tissues (n=10) by IHC. (D) USP2 protein levels in ten paired HCC-NT and HCC-T tissues as groups as well as (E) individual pairs. **P<0.01 and ***P<0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing, Staining, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2 mRNA expression levels were decreased in HCC-T tissues from HCC patients. (A) The expression levels of USP2 mRNA in human normal liver (n=12), HCC-NT (n=8) and HCC-T (n=21) detected by real-time PCR. (B) The expression levels of USP2 mRNA in paired HCC-NT and HCC-T tissues as groups as well as (C) individual pairs (n=8). *P<0.05 and **P<0.01 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Dysregulation of Usp2 mRNA expression in HCC-T tissues from HCC mice. A. The expression levels of Usp2 mRNA in mouse normal liver (n=16), HCC-NT (n=15) and HCC-T (n=15) samples. B. The expression levels of USP2 mRNA in paired HCC-T and HCC-NT tissues. **P<0.01 and ***P<0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2b is the predominant USP2 isoform in the liver. (A) The relative mRNA expression levels of USP2a, USP2b and USP2c in normal liver tissues from human (n=12) and (B) mice (n=16). **P<0.01 and ***P<0.001 in one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2b mRNA expression levels were markedly reduced in HCC-T tissues from HCC patients. (A) The expression levels of USP2 mRNA in human normal liver (n=12), HCC-NT (n=8) and HCC-T (n=21) detected by real-time PCR. (B) USP2b protein detected by Western blot in normal liver (n=8), HCC-NT (n=8) and HCC-T tissues (n=8). (C) Quantification of the USP2b protein levels in (B) by ImageJ. (D) The expression levels of USP2 mRNA in paired HCC-NT and HCC-T tissues as groups as well as (E) individual pairs (n=8). ***P<0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2b promoted cell proliferation. (A) HepG2 and (B) Huh 7 cells were transfected with USP2b or pcDNA vector, followed by monitoring cell growth for a period of 48 h by the MTS assays. (C) HepG2 and (D) Huh 7 cells were treated with increasing concentrations of USP2-specific inhibitor ML364 for 48 h, followed by monitoring cell proliferation by MTS assays. (E) HepG2 and (F) Huh 7 cells were transfected with USP2b or pcDNA vector in the presence or absence of USP2 inhibitor ML364 for 48 h, followed by monitoring cell proliferation by MTS assays. *P<0.05, **P<0.01, ***P<0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Transfection, Plasmid Preparation, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Knockout of USP2 significantly reduced cell proliferation. (A) Relative proliferation of parental HepG2 and USP2-KO (3A) and (B) USP2-KO (3E) cells over a period of 96 h. (C) USP2-KO (3A) and (D) USP2-KO (3E) cells were transfected with USP2b or pcDNA vector, followed by monitoring the cell proliferation over a period of 48 h. *P<0.05, **P<0.01 and ***P<0.001 in students’ t-test for pair-wise comparison.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Knock-Out, Transfection, Plasmid Preparation, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Identification of USP2-regulated target proteins with proteomic profiling. (A) Quantitative analysis of the proteomics dataset obtained from parental HepG2 (n=3 duplicates) and USP2-KO (3A) cells (n=3 duplicates) and (B) from HepG2 wt and USP2-KO (3E) cells (n=3 duplicates). Volcano plot shows the fold-change (1.5 fold up or down) versus p-value of protein abundance for the total of 4861 proteins identified. Red and green circles represent the significant (q<0.05) up- and down-regulated proteins in USP2-KO (3A) or USP2-KO (3E) compared with the parental HepG2 cells. (C) Overlap of the proteins differentially expressed in USP2-KO (3A) and USP2-KO (3E) cells when compared with the parental HepG2 cells. (D) The protein enrichment analysis of the overlapped altered proteins by KEGG and ShinyGo. (E) Overlap of upregulated proteins differentially expressed in USP2-KO (3A) and USP2-KO (3E) cells when compared with HepG2 cells. (F) The protein enrichment analysis of the overlapped upregulated proteins by KEGG and ShinyGo. (G) Overlap of downregulated proteins differentially expressed in USP2-KO (3A) and USP2-KO (3E) cells when compared with parental HepG2 cells. (H) The protein enrichment analysis of the overlapped downregulated proteins by KEGG and ShinyGo. (I) The heat map analysis of the USP2 regulated target proteins associated with cell proliferation, apoptosis, and tumorigenesis in the parental HepG2, USP2-KO (3A) and USP2-KO (3E) cells.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Quantitative Proteomics, Protein Enrichment

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Up- and down-regulated proteins in USP2-KO (3A) and USP2-KO (3E) cells when compared to parental HepG2 cells

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques:

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Confirmation of USP2-regulated target proteins by Western blotting. (A) Detection and quantification of USP2-regulated target protein STK4, (B) DPP4, (C) FABP1 and (D) EGFR in parental HepG2, USP2-KO (3A) and USP2-KO (3E) cells. ***P<0.001 with one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: E3 ligase HUWE1 promotes PDGF D-mediated osteoblastic differentiation of mesenchymal stem cells by effecting polyubiquitination of β-PDGFR

doi: 10.1016/j.jbc.2022.101981

Figure Lengend Snippet: Identification of the E3 ligase HUWE1 as an interactor of PDGF D-activated β-PDGFR in hBMSCs. A , immunoblot analysis of indicated proteins in hBMSC and BHPRS1 cells at indicated time points post treatments with 1 nM rPDGF D. B , immunoprecipitation as indicated, followed by LC–MS/MS analysis. Proteins that are significantly enriched in the immunoprecipitates of PDGF D-activated β-PDGFR as obtained from SFINX analysis are listed. C , the identified peptide sequences in the β-PDGFR and ubiquitin proteins in immunoprecipitates of β-PDGFR without (SFM) or with rPDGF D treatment are highlighted in yellow . The lysine residues with the Gly-Gly modification are marked in green . D , spectrum showing +114 Da at K860 of PDGF D-activated β-PDGFR. E , immunoblot analysis of immunoprecipitates from serum-starved hBMSCs treated with 1 nM rPDGF D using anti-β-PDGFR Ab or control IgG at indicated time points. F , USP2 catalytic domain (USP2CD)-mediated in vitro deubiquitination assay of immunoprecipitates of serum-starved hBMSCs treated without or with 1 nM rPDGF D for 10 min. G , immunoblot analysis of indicated proteins in immunoprecipitates from serum-starved hBMSCs treated without or with 1 nM rPDGF D using anti-HUWE1 Ab or control IgG. Total lysates (input) without or with rPDGF D treatment were also subjected to immunoblot analysis as a control. Ab, antibody; hBMSC, human bone marrow mesenchymal stem cell; HUWEI, HECT, UBA, and WWE domain–containing protein 1; IgG, immunoglobulin G; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; rPDGF, recombinant PDGF; USP2, ubiquitin-specific peptidase 2.

Article Snippet: Immunoprecipitates were washed with PBS, centrifuged, and incubated with 5 nM recombinant catalytic domain of USP2 (USP2CD; R&D Systems; caalog no.: E-504-050) at 37 ° C for 2 h in reaction buffer (50 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% glycerol) at pH 8.0 with periodic shaking.

Techniques: Western Blot, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, Modification, Control, In Vitro, Derivative Assay, Recombinant