Journal: Glia

Article Title: Impairment of protein degradation and proteasome function in hereditary neuropathies

doi: 10.1002/glia.23251

Figure Lengend Snippet: (A) The DUBs USP5, USP14 and UCH37 are increased on the 26S proteasome in S63del L. Proteasomes were incubated with HA-Ub-VS to modify the active site cysteines of the associated DUBs that copurified with them. Levels of modification, and thus activity of the DUB, were analyzed by western blot for HA and normalized to both the 20S ɑ subunits and the 19S subunit Rpt1. 5 HA-positive bands were detected and re-probing the blots with DUB-specific antibodies enabled the identification of Band 2 as USP5, Band 3 as USP14, and Band 4 as UCH37. The specific antibodies poorly recognized the HA-Ub-VS-bound DUBs, so primarily unmodified forms were detected. HA-positive bands 1 and 5 could not be identified with specific antibodies. The relative molecular weight of Band 1 is approximately ~140 kDa and antibodies against USP7 were tried. Band 5 is approximately 18 kDA and is potentially an HA-Ub-VS dimer.

Article Snippet: Proteins were transferred to either nitrocellulose (Protran; VWR) or PVDF (Immobilin FL; EMD Millipore) membranes and immunoblotting was performed using the following antibodies per manufacturer’s instructions: rabbit anti ubiquitin (VU-1) (LifeSensors); rabbit anti PSMA6, rabbit anti USP14, rabbit anti PSMD11 (Bethyl Laboratories); rabbit anti UCH37 (Abcam); rabbit anti USP5, rabbit anti PSMA7, rabbit anti PSMB5, rabbit anti RPT4, rabbit anti GADD34, mouse anti BAX (ProteinTech); goat anti USP14 (E-17), rabbit anti NRF1 (H-285), rabbit anti NRF2 (C-20), goat anti PSMC1 (C-16), rabbit anti ubiquitin (FL-76), rabbit anti PA200 (A-13), rabbit anti PA28beta (L-19) (Santa Cruz); rabbit anti ADRM1, mouse anti eIF2alpha, rabbit anti-phospho eIF2alpha, rabbit anti K48-ubiquitin, rabbit anti K63-ubiquitin (Cell Signaling); mouse anti α1234567 (MCP), mouse anti RPT5, mouse anti RPT1, rabbit anti RPN6 (Enzo); rabbit anti GRP78 (Novus); mouse anti PA28gamma, mouse anti VCP (BD Biosciences); mouse anti HA (Covance); chicken anti P0 (Aves).

Techniques: Incubation, Modification, Activity Assay, Western Blot, Molecular Weight

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) Schematic diagram showing the experimental procedure of the ICH rat. (b) Forelimb placing test and corner turn test of rats. (c) Brain water content of rats. (d) Real-time PCR and western blot were used to verify the expression of UBL4A in brain tissue. (e) Typical hematoxylin-eosin (HE) staining images presenting the striatal hematomas in rats. Scale bar: 500 µ m (left) and 100 µ m (right). (f) Fluoro-Jade B (FJB) staining was performed to detect neuronal cell death in rats. Scale bar: 50 µ m. (g) Co-localization of ubiquitin-like protein 4A (UBL4A) and NeuN was detected by immunofluorescence staining. Scale bar: 50 µ m. ICH, intracerebral hemorrhage. * P <0.05, ** P <0.01, ns: no significance.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining, Ubiquitin Proteomics, Immunofluorescence

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) The diagram depicts the flow chart of the cell experiment. (b) Real-time PCR and western blot were used to verify the expression level of ubiquitin-like protein 4A (UBL4A) in neurons. (c) CCK-8 assay was performed to determine cell viability. (d) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µ m. LV-shNC, negative control shRNA; LV-shUBL4A, shUBL4A knockdown; LV-vector, empty vector; LV-UBL4A, UBL4A overexpression. * P <0.05, ** P <0.01.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Ubiquitin Proteomics, CCK-8 Assay, TUNEL Assay, Staining, Negative Control, shRNA, Knockdown, Plasmid Preparation, Over Expression

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10 3 ). (b) Manganese superoxide dismutase (MnSOD) activity was measured with MnSOD assay kit. (c) Detection of mitochondrial membrane potential by JC-1 staining. (d) The relative mitochondrial DNA (mtDNA) level was detected by real-time PCR. LV-shNC, negative control shRNA; LV-shUBL4A, shUBL4A knockdown; LV-vector, empty vector; LV-UBL4A, UBL4A overexpression. * P <0.05, ** P <0.01.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Flow Cytometry, Activity Assay, Membrane, Staining, Real-time Polymerase Chain Reaction, Negative Control, shRNA, Knockdown, Plasmid Preparation, Over Expression

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) Real-time PCR to verify the expression of miR-34a-5p in neurons. (b) Real-time PCR and western blot were used to verify the expression level of ubiquitin-like protein 4A (UBL4A) in neurons with miR-34a-5p knockdown. (c) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µ m. (d) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10 3 ). LV-shNC, negative control shRNA; LV-miR-34a-sponge, miR-34a-5p knockdown. ** P <0.01.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Ubiquitin Proteomics, Knockdown, TUNEL Assay, Staining, Flow Cytometry, Negative Control, shRNA

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) Predicted miR-34a-5p target sequences in 3′-UTR of Ubl4a (Left). Dual-luciferase reporter assay was conducted to verify whether ubiquitin-like protein 4A (UBL4A) was the target of miR-34a-5p (Right). NC mimics, negative control mimics; WT, wild type; MUT, mutant type. (b) Western blot was used to verify the expression level of UBL4A in neurons. (c) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µ m. (d) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10 3 ). LV-shNC, negative control shRNA; LV-miR-34a-sponge, miR-34a-5p knockdown. LV-shUBL4A, shUBL4A knockdown. * P <0.05, ** P <0.01.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Luciferase, Reporter Assay, Ubiquitin Proteomics, Negative Control, Mutagenesis, Western Blot, Expressing, TUNEL Assay, Staining, Flow Cytometry, shRNA, Knockdown

Journal: Journal of cellular biochemistry

Article Title: Computational and Biochemical Studies of Isothiocyanates as Inhibitors of Proteasomal Cysteine Deubiquitinases in Human Cancer Cells

doi: 10.1002/jcb.27157

Figure Lengend Snippet: Determination of interactions of BITC, PEITC and SFN with 19S-associated DUBs USP14 and UCHL5 by computational docking. Structures of BITC, PEITC and SFN are shown at the top. Docking studies were conducted between USP14 (A–C) and UCHL5 (D–F) with BITC, PEITC, and SFN (left, middle and right columns, respectively). The generated conformations (docking modes) between the active site amino acid residues of USP14 or UCHL5 and the ligands (ITCs) have been displayed. The free energy scores of the reactions of BITC, PEITC and SFN with USP14 were −3.5, −3.7 and −3.0 kcal/mol, respectively, while that with UCHL5 were −5.0, −5.0 and −3.9 kcal/mol, respectively.

Article Snippet: UCHL5 polyclonal antibody, PSMD14/POH1 polyclonal antibody, and HRP-conjugated beta Actin monoclonal antibody were all purchased from Proteintech™.

Techniques: Generated

Journal: Journal of cellular biochemistry

Article Title: Computational and Biochemical Studies of Isothiocyanates as Inhibitors of Proteasomal Cysteine Deubiquitinases in Human Cancer Cells

doi: 10.1002/jcb.27157

Figure Lengend Snippet: Inhibition of active site-directed labeling of proteasomal DUBs by BITC, PEITC and SFN. Breast cancer MDA-MB-231 cells were either not treated (NT) or treated for 3 h with either DMSO, PEITC, or BITC at the indicated concentrations (A), or SFN at 25 µM (B). Prostate cancer 22Rv1 cells were either not treated (NT) or treated for 3 h with DMSO (DM) or BITC at the indicated concentrations (C), or 20 µM of the indicated ITC (D, E). After that, 25 µg cell lysates were subsequently labelled with Ub-VS (1 µM) in vitro for 30 min at 37°C followed by SDS gel electrophoresis and immunoblotting with USP14 and UCHL5 antibodies to evaluate the inhibitory activity of these ITCs on the 19S-associated deubiquitinases USP14 and UCHL5. The upper bands represent the active USP14 or UCHL5 bound by Ub-VS (USP14-Ub-VS and UCHL5-Ub-VS conjugates), while the lower bands are unbound USP14 or UCHL5.

Article Snippet: UCHL5 polyclonal antibody, PSMD14/POH1 polyclonal antibody, and HRP-conjugated beta Actin monoclonal antibody were all purchased from Proteintech™.

Techniques: Inhibition, Labeling, In Vitro, SDS-Gel, Electrophoresis, Western Blot, Activity Assay

Journal: Journal of cellular biochemistry

Article Title: Computational and Biochemical Studies of Isothiocyanates as Inhibitors of Proteasomal Cysteine Deubiquitinases in Human Cancer Cells

doi: 10.1002/jcb.27157

Figure Lengend Snippet: Effects of ITCs on levels of ubiquitinated proteins, apoptosis and 19S-DUBs in breast cancer MDA-MB-468 and MDA-MB-231 cells. MDA-MB-468 and MDA-MB-231 cells were either treated with DMSO (DM) or 25 µM of BITC (A), PEITC (B) or SFN (C) for the indicated horus (h). The cells were harvested, and protein lysates were extracted for electrophoresis, immunoblotting for ubiquitinated proteins (Ub-Prs, with anti-ubiquitin), PARP, USP14, UCHL5, and RPN11 as well as β-actin (as an internal control for protein loading and normalization between samples).

Article Snippet: UCHL5 polyclonal antibody, PSMD14/POH1 polyclonal antibody, and HRP-conjugated beta Actin monoclonal antibody were all purchased from Proteintech™.

Techniques: Electrophoresis, Western Blot

Journal: Journal of cellular biochemistry

Article Title: Computational and Biochemical Studies of Isothiocyanates as Inhibitors of Proteasomal Cysteine Deubiquitinases in Human Cancer Cells

doi: 10.1002/jcb.27157

Figure Lengend Snippet: Effects of ITCs on levels of ubiquitinated proteins, apoptosis and 19S-DUBs in prostate cancer 22Rv1 cells. 22Rv1 cells were treated with DMSO (DM) or 25 µM of BITC (A) or SFN (B) for the indicated hours (h), followed by Western blotting for ubiquitinated proteins (Ub-Prs), PARP, USP14, UCHL5 and RPN11 as well as β-actin (as an internal control).

Article Snippet: UCHL5 polyclonal antibody, PSMD14/POH1 polyclonal antibody, and HRP-conjugated beta Actin monoclonal antibody were all purchased from Proteintech™.

Techniques: Western Blot

Journal: The Journal of Experimental Medicine

Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes

doi: 10.1084/jem.20242474

Figure Lengend Snippet: Intracellular portion of FCRL3 interacts with UBASH3A. (A) Schematic representation of the Protein A-TurboID experimental workflow. An anti-FCRL3 antibody was added to FCRL3-expressing or nonexpressing Jurkat cells followed by the addition of Protein A-TurboID fusion protein and biotin. After biotinylation and extensive washing, biotinylated proteins were recovered using streptavidin-conjugated beads and subjected to mass spectrometry analysis. (B) Example of Protein A-TurboID experiment. After incubation of FCRL3-expressing Jurkat cells (or empty vector control cells) with a mouse monoclonal anti-FCRL3 antibody together with recombinant Protein A-TurboID, biotinylated proteins were recovered by streptavidin pull-down, followed by western blot to confirm enrichment of FCRL3. The antibody used for immunoblot was a rabbit polyclonal anti-FCRL3. (C) Differentially retrieved proteins following FCRL3 Protein A-TurboID and mass spectrometry of FCRL3-expressing versus nonexpressing cells (log 2 FC ≥ |2|; P ≤0.01; N = 5 replicates). (D) Example of FCRL3 IP. Jurkat cells expressing FCRL3 (or empty vector control) were lysed, and 1.5 mg of protein extract was used for IP with a rabbit polyclonal anti-FCRL3 antibody, followed by western blot with the same antibody. Two independent representative FCRL3 IPs are shown. (E) Differentially retrieved proteins following FCRL3 IP-MS of FCRL3-expressing versus nonexpressing cells (log 2 FC ≥ |2|; P ≤0.01; N = 4 replicates). (F) Co-IP of FCRL3 with UBASH3A. HEK cells were transfected with the indicated plasmids, followed by IP of FCRL3 using a rabbit polyclonal anti-FCRL3 antibody and immunoblot for either UBASH3A (top) or FCRL3 itself (bottom). Data are representative of N = 2 independent experiments. (G) Schematic representation of the FCRL3 protein and C-terminal truncations (top). All proteins were efficiently expressed, as shown by western blot (bottom). (H) Heatmap showing the differentially enriched proteins by IP-MS of Jurkat cells transduced with full-length FCRL3 and its truncations. N = 3–4 independent samples. Significant differences between multiple experimental conditions were assessed with an ANOVA multiple-sample test (0.01 permutation-based FDR cut-off, 250 randomizations). Significant proteins were filtered, and Z-score normalization was applied to each protein across all samples. Unsupervised hierarchical clustering was employed to visualize on a heatmap proteins with a positive Z-score value for all replicates of the conditions LV-FCRL3 and LV-FCRL3-93 aa. IP, immunoprecipitation. Data underlying this figure can be found in . Source data are available for this figure: .

Article Snippet: The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.

Techniques: Expressing, Mass Spectrometry, Incubation, Plasmid Preparation, Control, Recombinant, Western Blot, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Transduction, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes

doi: 10.1084/jem.20242474

Figure Lengend Snippet: IP-MS and TurboID-MS confirm the interaction between FCRL3 and UBASH3A. (A) Schematic representation of FCRL3 and the C-terminally tagged FCRL3-3xFLAG protein. The 3xFLAG tag is shown as green squares. (B) Surface (left) FCRL3 staining and intracellular (right) anti-FLAG staining showing surface expression of the FCRL3-3xFLAG construct. (C) Western blot showing efficient immunoprecipitation of FCRL3-3xFLAG with anti-FLAG agarose beads in Jurkat cells expressing LV-FCRL3 and LV-FCRL3-3xFLAG. Immunoblot was performed using an anti-FCRL3 antibody. (D) Scatter plot showing the enriched proteins in the IP-MS of the FCRL3-3xFLAG compared with untagged FCRL3. In this experiment, to enhance the separation of true interactors from experimental noise, an additional filter was applied by selecting proteins present in at least 3 out of 4 replicates of the FCRL3-3xFLAG condition and in <2 replicates of the untagged control (FCRL3), followed by imputation of the missing values. A two-sided two-samples t test (0.01 permutation-based FDR cut-off, 250 randomizations) was employed to identify significant changes, and the results were visualized with a volcano plot generated with R, version 4.4.2. (E) Schematic representation of the Protein A-TurboID experimental workflow for UBASH3A. The anti-UBASH3A antibody was added to Jurkat cells ectopically expressing FCRL3 or the EGFR-FCRL3 140 aa chimera (or control cells), followed by the addition of Protein A-TurboID fusion protein and biotin. After streptavidin pull-down, biotinylated proteins were analyzed by mass spectrometry. (F) Differentially enriched proteins in the FCRL3-transduced cells versus control (left) and chimera-transduced cells versus control (right). N = 4 independent samples. Source data are available for this figure: .

Article Snippet: The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.

Techniques: Protein-Protein interactions, Staining, Expressing, Construct, Western Blot, Immunoprecipitation, Control, Generated, Mass Spectrometry