usp2 catalytic domain  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: CAPN15 is a non-proteasomal, ubiquitin-directed calpain protease that regulates cell adhesion by cleaving E-cadherin

doi: 10.1016/j.jbc.2025.111034

Figure Lengend Snippet: CAPN15 recognizes the ubiquitinated cadherin-catenin complex via the NZF domains. A , differential interference contrast images of HCT116, KO, WTtg, CStg, and NEtg cells. Scale bar, 20 μm. B , the mean log 2 fold change of the protein abundance (CStg versus NEtg immunoprecipitates) with -log 10 p value of the interactome was shown on the x- and y-axes, respectively. Significantly increased proteins were defined as those with the value of the log 2 fold change >1 with p value < 0.05 (plots in a colored area). It should be noted that there was no change in abundance of CAPN15 in the CStg and NEtg immunoprecipitates. C , the heatmap shows the relative abundance of cadherins and catenins in the KO, WTtg, CStg, and NEtg immunoprecipitates. D , KO, WTtg, CStg, and NEtg cells were treated with dimethyl sulfoxide (DMSO; vehicle) or 1 μM MLN7243. Cell lysates were subjected to immunoprecipitation using an anti-FLAG antibody, followed by western blotting. Asterisks indicate nonspecific bands. Vertical lines indicate the ubiquitinated forms. E , immunoprecipitated fraction from DMSO (vehicle)-treated CStg cells was incubated with or without ubiquitin-specific protease 2 (USP2cc), followed by western blotting. The asterisk indicates the non-specific band. Vertical lines indicate the ubiquitinated forms.

Article Snippet: Anti-FLAG immunoprecipitate prepared from CStg cells was incubated with 1 μM USP2 catalytic domain (USP2cc; R&D Systems) for 1 h at 37 °C.

Techniques: Quantitative Proteomics, Immunoprecipitation, Western Blot, Incubation, Ubiquitin Proteomics

Journal: Journal of Virology

Article Title: The vaccinia virus protein, C16, promotes the ubiquitylation and relocalization of the antiviral E3 ubiquitin-ligase, TRIM25

doi: 10.1128/jvi.00898-25

Figure Lengend Snippet: TRIM25 was ubiquitylated in VACV-Cop-infected cells. (A) Cell lysates from uninfected HeLa cells or HeLa cells infected for 4 h with VACV-Cop (MOI of 10) were used to perform IPs with an anti-conjugated Ub Ab, and IPs were then treated with (+) or without (−) the USP2 deubiquitinase before being immunoblotted for TRIM25 (upper panel) or conjugated Ub. HC = heavy chain of immunoprecipitating Ab. (B and C) Lysates were prepared from HeLa cells treated with the indicated concentrations of MLN4924 and either left uninfected or infected with VACV-Cop for 4 h (MOI of 10). Lysates were then immunoblotted with Abs against the indicated proteins. The anti-Cul1 Ab recognizes both unmodified Cul1 and NEDD8-modified (Cul1 NEDD8 ). (D and E) Lysates from uninfected or VACV-Cop-infected (4 h; MOI of 10) HeLa cells or HeLa cells lacking ISG15 were immunoblotted with Abs against the indicated proteins. The two ISG15 knock-out clones (ISG #1 and #2) were generated using different crRNAs. Molecular mass markers are indicated to the left of blots.

Article Snippet: Samples from anti-conjugated Ub IP were washed and incubated in deubiquitylation buffer (50 mM NaCl, 50 mM Tris, pH 7.4, and 50 mM DTT) with 10 μM USP2 protein (R&D systems, #E-504) at 30°C for 2 h. To stop the reaction, samples were boiled with sample buffer for 10 minutes.

Techniques: Infection, Modification, Knock-Out, Clone Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: A versatile fluorescence polarization-based deubiquitination assay using an isopeptide bond substrate mimetic (IsoMim)

doi: 10.1016/j.jbc.2025.110342

Figure Lengend Snippet: Fluorescence polarization-based IsoMim assay principle and optimization. A , crystal structure of the catalytic core domain of USP11, depicted as a grey surface representation, in complex with Ub-GGG (in purple cartoon representation with the GGG extension colored in black ; PDB code: 8OYP ) B , schematic depiction of the probe design and assay principle. Representation of the DiUb 3G -FM substrate with the predicted Ub L73P -Ub structure (Alphafold ) in cartoon representation shown in purple and the C-terminal GGGC extension labeled with Fluorescein-5-maleimide (FM) shown as a chemical structure. The C-terminal ubiquitin moiety, the P1 position according to canonical protease nomenclature, is boxed and shaded in purple and forms the basis of a Ub 3G -FM probe. The GGGC extension is highlighted by a black-rimmed box . Upon DUB cleavage of DiUb 3G -FM, the rotational freedom of the liberated GGGC-FM moiety increases, leading to depolarization of the incident polarized light and a decrease in FP signal. C , SDS-PAGE gel analysis comparing mono-ubiquitin with the generated substrate DiUb 3G -FM, in the absence and presence of 50 nM USP2 after 30 min incubation, and unlabeled DiUb 3G . D , comparison of the FP signal from DiUb 3G -FM (10 nM) with free fluorescein (10 nM) demonstrating the assay window. E , converted curves for 10 nM USP2, USP2 C276A and heat inactivated USP2 (USP ia ) showing the FP change. F , representative progress curves for USP2, USP2 C276A and heat inactivated USP2 (USP ia ) in comparison with the probe alone control ( top inset ). Rates of reaction at three different USP2 concentrations with 10 nM DiUb 3G -FM ( bottom ). Data points represent the mean ± SD of three independent experiments.

Article Snippet: The USP2 catalytic domain expression construct as used for the structure determination of a covalent Ubiquitin-USP2 complex (PDB code: 2IBI ) was a gift from Cheryl Arrowsmith (Addgene plasmid # 36894; http://n2t.net/addgene:36894 ; RRID: Addgene 36,894).

Techniques: Fluorescence, Labeling, Ubiquitin Proteomics, SDS Page, Generated, Incubation, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: A versatile fluorescence polarization-based deubiquitination assay using an isopeptide bond substrate mimetic (IsoMim)

doi: 10.1016/j.jbc.2025.110342

Figure Lengend Snippet: Dose response curves of different DUBs and PR-619 as the inhibitor. A , dose–response curves for inhibition by pan-inhibitor PR-619 for the catalytic core domains of USP4, USP15, USP2 and UCHL3. Data were fitted using non-linear regression in GraphPad Prism, and data points show mean ± SD from three independent experiments. B , pIC50 values of PR-619 DUB inhibition (error = SE; n = 3). C , proof-of principle high-throughput screening results using USP4-D1D2 and PR-619 in a 384-well plate layout. The DiUb 3G -FM reagent alone and selected wells together with USP4-D1D2 served as control groups, measured as quadruplicates on the plate as indicated in . PR-619 was added in random places in quadruplicate (plate layout displayed in ). Highlighted in light green are the data from wells where USP4-D1D2 was incubated with PR-619. Dashed lines indicate 0% and 50% of inhibition.

Article Snippet: The USP2 catalytic domain expression construct as used for the structure determination of a covalent Ubiquitin-USP2 complex (PDB code: 2IBI ) was a gift from Cheryl Arrowsmith (Addgene plasmid # 36894; http://n2t.net/addgene:36894 ; RRID: Addgene 36,894).

Techniques: Inhibition, High Throughput Screening Assay, Control, Incubation