Journal: bioRxiv

Article Title: A genetic screen to identify deubiquitinases as regulators of IRF7

doi: 10.1101/2025.09.09.675186

Figure Lengend Snippet: (A) RAW-Luci macrophages were either infected with SeV, or transfected with poly(I:C) (pIC) using Lipofectamine (LF), or treated with pIC alone. Otud5 mRNA levels were quantified by qRT-PCR. (B–G) RAW264.7 cells were transfected with NT, Otud5 (O5), or Usp2 (U2) siRNAs, infected with SeV for 8 hours, and mRNA levels of Otud5, Usp2, Ifna, Ifnb1, Ifit1, and Ifit3 were analyzed by qRT-PCR. (H–J) HEK-KO.IRF7 cells were transfected with NT, OTUD5 (O5), or USP2 (U2) siRNAs and infected with SeV. USP2 , OTUD5 , and IFNA mRNA levels were measured by qRT-PCR. The data represent mean ± SEM (A-G, J), * p<0.05, ** p<0.001, *** p<0.001, **** p<0.0001.

Article Snippet: Plasmids included HA-Ub-K27O (Addgene #22903), HA-Ub-K63O (Addgene #17606), OTUD5 (Sino Biologicals #HG22452-CF), and USP2 (Sino Biologicals #HG12713-UT).

Techniques: Infection, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A genetic screen to identify deubiquitinases as regulators of IRF7

doi: 10.1101/2025.09.09.675186

Figure Lengend Snippet: (A) USP2:IRF7 interaction in HEK-KO.IRF7 cells was assessed by co-immunoprecipitation at the indicated times post-SeV infection. (B–C) Proximity ligation assay (PLA) was performed in WT primary BMDMs using anti-USP2 and anti-IRF7 antibodies in mock or SeV-infected (8 hpi) cells. Red dots represent interaction signals, quantified in (C). (D) OTUD5:IRF7 interaction in HEK-KO.IRF7 cells was analyzed by co-immunoprecipitation following SeV infection. (E–F) PLA was conducted in WT BMDMs using anti-OTUD5 and anti-IRF7 antibodies in mock or SeV-infected cells. Interaction signals were quantified in (F).

Article Snippet: Plasmids included HA-Ub-K27O (Addgene #22903), HA-Ub-K63O (Addgene #17606), OTUD5 (Sino Biologicals #HG22452-CF), and USP2 (Sino Biologicals #HG12713-UT).

Techniques: Immunoprecipitation, Infection, Proximity Ligation Assay

Journal: bioRxiv

Article Title: A genetic screen to identify deubiquitinases as regulators of IRF7

doi: 10.1101/2025.09.09.675186

Figure Lengend Snippet: (A) HEK-KO.IRF7 cells were transfected with Flag.OTUD5 or USP2 plasmids, and the cell lysates were analyzed by immunoblot, as indicated. (B–D) HEK-KO.IRF7 cells were transfected with HA-tagged Ub-K63O, Ub-K27O, or Ub-K33O plasmids, in the absence or the presence of OTUD5 or USP2, as indicated, and infected with SeV. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-HA, as indicated. (E–G) Full-length and truncated IRF7 mutants (E) were co-transfected with Ub-K63O or Ub-K27O, followed by SeV infection. Ub-IRF7 levels were measured by IP and immunoblot (F) and quantified using ImageJ (G). (H) Schematic of IRF7 protein domains and putative ubiquitin linkage sites (K63 and K27); DBD, DNA-binding domain. (I) Cells were transfected with HA.Ub-K27O and infected with SeV. Ub-IRF7 was analyzed as in (A). (J) IRF7-WT or KR mutants (K1: K327R, K2: K329R, K3: K327/329RR) were co-transfected with Ub-K27O, infected with SeV, and analyzed for Ub-IRF7. The lower panel shows the expression of the IRF7 mutants. EV, empty vector; Ub-IRF7, ubiquitinated IRF7.

Article Snippet: Plasmids included HA-Ub-K27O (Addgene #22903), HA-Ub-K63O (Addgene #17606), OTUD5 (Sino Biologicals #HG22452-CF), and USP2 (Sino Biologicals #HG12713-UT).

Techniques: Transfection, Western Blot, Infection, Immunoprecipitation, Ubiquitin Proteomics, Binding Assay, Expressing, Plasmid Preparation

Journal: The EMBO Journal

Article Title: Dual roles of HSP70 chaperone HSPA1 in quality control of nascent and newly synthesized proteins

doi: 10.15252/embj.2020106183

Figure Lengend Snippet: Cell lysates were prepared from MCF7 cells maintained at 37°C or heat‐shocked at 42°C for 1 h. Where indicated, lysate from heat‐shocked cells was incubated with recombinant USP2 to remove polyubiquitin chains. Equal amounts of cell lysate were assayed for the levels of the indicated proteins by immunoblotting. A quantification of the levels of K48‐linked polyubiquitin is shown in the bar graph (means ± SD, n = 4, number indicates P value calculated with the two‐stage linear step‐up procedure of Benjamini, Krieger, and Yekutieli). Equal amounts of cell lysate described in (A) were fractionated by sucrose density gradient centrifugation, and K48‐linked polyubiquitin was detected by immunoblotting. Quantification of K48‐linked polyubiquitin across the gradients relative to the levels obtained in MCF7 cells maintained at 37°C (means ± SEM, n = 3). Trypsin‐like proteasome activity determined in the same fractions as in (B). Results are shown relative to the proteasome activity obtained in MCF7 cells maintained at 37°C (means ± SEM, n = 3). Source data are available online for this figure.

Article Snippet: To digest K48‐polyubiquitin chains, the lysate was incubated with 7 μg of recombinant human USP2 catalytic domain protein (Boston Biochem, E‐506) for 1 h at 25°C.

Techniques: Incubation, Recombinant, Western Blot, Gradient Centrifugation, Activity Assay

Journal: The EMBO Journal

Article Title: Dual roles of HSP70 chaperone HSPA1 in quality control of nascent and newly synthesized proteins

doi: 10.15252/embj.2020106183

Figure Lengend Snippet:

Article Snippet: To digest K48‐polyubiquitin chains, the lysate was incubated with 7 μg of recombinant human USP2 catalytic domain protein (Boston Biochem, E‐506) for 1 h at 25°C.

Techniques: Recombinant, Sequencing, Magnetic Beads, Staining, Protease Inhibitor, DNA Purification, Bicinchoninic Acid Protein Assay, Modification, Software, Imaging

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 cell biology

Article Title: Hrd1 forms the retrotranslocation pore regulated by auto-ubiquitination and binding of misfolded proteins.

doi: 10.1038/s41556-020-0473-4

Figure Lengend Snippet: Fig. 1 | Electrophysiological characterization of Hrd1 gating dynamics following auto-ubiquitination. a, The current recordings for PLBs after the addition of liposomes containing non-ubiquitinated Hrd1 at the indicated constant holding potentials (left) and the corresponding point histograms (right). b, Current recordings as in a, but with polyubiquitinated Hrd1 liposomes. The numbers in the zoomed-in plot indicate various conductance states. c, Liposomes containing fluorescently labelled Hrd1 were incubated with ubiquitination mix with or without ATP. Samples from the indicated time points were analysed by SDS–polyacrylamide gel electrophoresis (PAGE) and fluorescence scanning. d, Fusion rates of the PLBs with Hrd1 or Ubc6-containing liposomes treated with the indicated conditions. For the quantifications, proteoliposomes were repeatedly added to bilayers and channel fusion events were detected over multiple hours. e, A conductance-state histogram of ubiquitinated Hrd1 as calculated from gating transitions that were recorded at varying membrane potentials. f, Current recordings of ubiquitinated Hrd1 before (left) and after (right) the addition of 1 µM Usp2 to the cis side at the indicated voltages. The closed and various open states of the channels are indicated by c, closed state and ox, open states. g, The probability of ubiquitinated Hrd1 channels being open before and after side-specific deubiquitination. In a–c,f, representative samples of three independent experiments are shown. In d,g, the mean ± s.e.m. (n = 3 independent experiments) are shown. Source data and unprocessed gels are provided.

Article Snippet: Usp2, with an N-terminal His6 tag in the pET28a vector, was a gift from C. Arrowsmith (Addgene plasmid no. 36894).

Techniques: Ubiquitin Proteomics, Liposomes, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence, Membrane

Journal: Nature cell biology

Article Title: Hrd1 forms the retrotranslocation pore regulated by auto-ubiquitination and binding of misfolded proteins.

doi: 10.1038/s41556-020-0473-4

Figure Lengend Snippet: Fig. 3 | Binding of CPY* to the luminal and cytosolic sides of Hrd1. a, Bead-immobilized fluorescently labelled Hrd1 (250 nM) in liposomes was incubated with ubiquitination mix with or without ATP, followed by incubation with fluorescently labelled CPY* (50 nM). Where indicated, Usp2 incubation was performed before incubation with CPY*. The samples were analysed by SDS–PAGE and fluorescence scanning. b, The quantification of four experiments from a. The mean ± s.d (n = 4 independent experiments) are shown. c, Experiments performed as in a, but with 20 nM of either CPY* or CPY WT and increasing Hrd1 concentrations. The bound fraction was quantified from supernatants and normalized to bead-only controls. The mean ± s.d. (n = 4 independent experiments) are shown. d, Fluorescently labelled Hrd1 (200 nM) in nanodiscs was incubated with ubiquitination mix with or without ATP. Samples from the indicated time points were analysed by SDS–PAGE and fluorescence scanning. e, Fluorescently labelled CPY* or CPY WT (20 nM) was incubated with bead-immobilized fluorescently labelled Hrd1 in nanodiscs that had been treated with ubiquitination mix with or without ATP. The quantification was performed as in b. The mean ± s.d. (n = 3 independent experiments) are shown. f, Fluorescently labelled CPY* or CPY WT (50 nM) was incubated with either empty nanodiscs or Hrd1 nanodiscs (at the concentrations indicated) that had been treated with ubiquitination mix with or without ATP; fluorescence anisotropy was measured. The left and right axes indicate anisotropy changes in the absence and presence of ATP, respectively. The top axis indicates approximate scaffold concentrations. CPY* + ATP: n = 4 independent experiments; CPY* − ATP: n = 5 independent experiments; CPY WT: n = 3 independent experiments; empty nanodiscs: n = 2 independent experiments. g, Fluorescently labelled CPY* (100 nM) was incubated with fluorescently labelled Hrd1 (200 nM) in nanodiscs and ubiquitination mix with or without ATP. Samples from indicated time points were analysed as before. h, The quantification of the ubiquitination experiments performed in d,g. The mean ± s.d. (n = 3 independent experiments) are shown. Source data and unprocessed gels are provided.

Article Snippet: Usp2, with an N-terminal His6 tag in the pET28a vector, was a gift from C. Arrowsmith (Addgene plasmid no. 36894).

Techniques: Binding Assay, Liposomes, Incubation, Ubiquitin Proteomics, SDS Page, Fluorescence

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Ubiquitination-Deubiquitination Balance Dictates Ligand-stimulated PTHR Sorting

doi: 10.1002/jbmr.494

Figure Lengend Snippet: (A) ROS cells were transiently transfected with HA-PTHR and treated with 100 nM PTH(1–34) or 1 μM PTH(7–34) for 1 hour. USP2 expression was assayed by immunoblot. (B) ROS cells were transfected with HA-USP2 and Flag-PTHR. After 48 hours, cells were treated with agonist as indicated. PTHR was detected using an anti-Flag primary antibody (1:1000). Values are mean ± SEM from ≥ 3 independent experiments. *p<0.05 vs. PTH(7–34).

Article Snippet: Polyclonal anti-EPS15 antibody, EPS15 shRNA and USP2 shRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Expressing, Western Blot

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Ubiquitination-Deubiquitination Balance Dictates Ligand-stimulated PTHR Sorting

doi: 10.1002/jbmr.494

Figure Lengend Snippet: ROS cells were transfected with HA-PTHR. After 24 hours cells were transfected with shRNA-USP2 and incubated for 48 hours. Cells were then treated with 100 nM PTH(1–34) for 30 or 120 minutes. Total lysates and immunoprecipitated protein were analyzed by SDS-polyacrylamide gels and transferred to Immobilon-P membranes. Representative autoradiograms of USP2 (A) Ubiquitinated PTHR (B) and total PTHR (C) are shown. Values are mean ± SEM from ≥ 3 independent experiments. *p<0.05; **p < 0.01 vs. corresponding scrambled shRNA value.

Article Snippet: Polyclonal anti-EPS15 antibody, EPS15 shRNA and USP2 shRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, shRNA, Incubation, Immunoprecipitation