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

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Boxplot of the UCH-L1 mRNA expression levels in four different subtypes of BRCA from the GEPIA2 database. B mRNA expression levels of UCH-L1 in TNBC and normal breast tissues from the GEPIA2 database. C IHC analysis of TNBC ( n = 40) and normal breast tissue samples ( n = 20) was performed using anti-UCH-L1 antibody. Representative images of IHC staining and quantified data presented in box plots are shown. Scale bar, 100 μm. D Western blot analysis of UCH-L1 protein expression level in normal breast cell and multiple TNBC cell lines. β-Actin was used as a loading control. E Kaplan-Meier plots for the overall survival of TNBC patients according to UCH-L1 mRNA expression from the Kaplan–Meier Plotter database. F Kaplan-Meier plots for the relapse-free survival of TNBC patients according to UCH-L1 protein expression from the Kaplan–Meier Plotter database. G Kaplan-Meier plots for the overall survival of chemotherapy-treated TNBC patients according to UCH-L1 mRNA expression from the Kaplan–Meier Plotter database. H The receiver operating characteristic (ROC) curve between UCH-L1 expression and therapeutic responses to taxane-containing NAC in breast cancer cohorts. I The correlation analysis between the efficacy of taxane-based chemotherapy and UCH-L1 expression. Chi-square test was performed to calculate statistical significance between two comparator groups. Stacked bar chart showed the proportion of patients with or without response in high or low UCH-L1 groups. * p < 0.05; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Expressing, Immunohistochemistry, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Knockdown efficiency of UCH-L1 in TNBC cells was evaluated by Western blot assay. B HCC1806 and BT549 cells with or without UCH-L1 depletion were incubated with indicated concentrations of PTX for 72 h, and cell viability was measured by CCK-8 assay. C Colony formation of HCC1806 and BT549 cells (500 cells per well) with or without UCH-L1 depletion in the presence of DMSO or 20 nM PTX. D HCC1806 and BT549 cells with or without UCH-L1 depletion were incubated with 20 nM PTX for 24 h, and cell proliferation was measured by EdU assay. Scale bars, 150μm. E HCC1806 and BT549 cells with or without UCH-L1 depletion were incubated with 20 nM PTX for 48 h, and cell apoptosis was measured by TUNEL assay. Scale bars, 150μm. F A schematic view of the animal study plan ( n = 6). G Curves of tumor growth. H Photopraphs of mice tumors of each group at the termination of the experiment. I Tumor weights were measured at the end of the experiment. J Characterization of HCC1806 xenograft tumors with histologic analysis by IHC staining of Ki67 and TUNEL. K The effect of treatment on mice body weight. Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Knockdown, Western Blot, Incubation, CCK-8 Assay, EdU Assay, TUNEL Assay, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Volcano plot of quantitative proteomics showed the differentially expressed genes between UCH-L1 knockdown and control HCC1806 cells. B KEGG pathway analysis for differentially expressed proteins in HCC1806 cells with or without UCH-L1 knockdown. C Effect of UCH-L1 knockdown on the ECAR of HCC1806 cells was detected using the Seahorse analyzer. D Quantification analysis of glycolysis, glycolytic capacity and glycolytic reserve levels in the ECAR assays. E - G Glucose consumption, lactate and ATP production were determined in control and UCH-L1 knockdown HCC1806 and BT549 cells. H - J Glucose consumption, lactate and ATP production were determined in MDA-MB-231 cells with or without UCH-L1 overexpression. K - L Colony formation of MDA-MB-231 cells with or without UCH-L1 overexpression in the presence of DMSO or PTX and 2-DG. M MDA-MB-231 cells from the indicated groups were incubated with PTX for 72 h, and cell viability was measured by CCK-8 assay. N MDA-MB-231 cells stably expressing empty vector (EV) or flag-tagged UCH-L1 were incubated with DMSO or PTX and 2-DG for 24 h, and cell proliferation was measured using EdU. Scale bars, 150 μm. O MDA-MB-231 cells with or without UCH-L1 overexpression were incubated with DMSO or PTX and 2-DG, and cell apoptosis was measured using TUNEL assay. Scale bars, 100 μm. Results shown are representative of three independent experiments. Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Quantitative Proteomics, Knockdown, Control, Over Expression, Incubation, CCK-8 Assay, Stable Transfection, Expressing, Plasmid Preparation, TUNEL Assay

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A The top twenty UCH-L1-interacting proteins according to the number of identified unique peptides by Co-IP-MS assay. B HEK293T cells were transfected with Flag-PKM2 and Myc-UCH-L1 plasmids, and then subjected to immunoprecipitation with anti-Flag or anti-Myc antibodies. The lysates and immunoprecipitates were then blotted. C Immunoprecipitation analysis with the indicated antibodies was performed to detect endogenous UCH-L1 and PKM2 interaction in BT549 cells. D Immunofluorescence assay to confirm the colocalization of UCH-L1 and PKM2 in HCC1806 cells. E Purified recombinant GST-UCH-L1 interacts with PKM2. GST-UCH-L1 and GST proteins were pulled down with glutathione beads. PKM2 was detected by Western blot. F Schematic representation of full-length (FL) UCH-L1, PKM2 and different truncation mutants. G Myc-UCH-L1 FL or indicated truncation mutants were co-expressed with Flag-PKM2 in HEK293T cells. Flag-PKM2 was immunoprecipitated with anti-Flag antibody, followed by immunoblotting analysis of Myc-UCH-L1 using Myc antibody. H Flag-PKM2 FL or indicated truncation mutants were co-expressed with Myc-UCH-L1 in HEK293T cells. Myc-UCH-L1 was immunoprecipitated with anti-Myc antibody, followed by immunoblotting analysis of Flag-PKM2 using Flag antibody. I MDA-MB-231 cells transfected with the indicated plasmid were incubated with PTX for 72 h, and cell viability was measured by CCK-8 assay. J , K MDA-MB-231 cells with UCH-L1 overexpression were transfected with PKM2 siRNA, and cell viability was detected by CCK-8 assay after 72 h incubation of the indicated concentrations of PTX. Results shown are representative of three independent experiments. Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Immunofluorescence, Purification, Recombinant, Western Blot, Plasmid Preparation, Incubation, CCK-8 Assay, Over Expression

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Immunoblotting analysis of PKM2 and UCH-L1 in UCH-L1 knockdown TNBC cells. B Immunoblotting analysis of PKM2 and UCH-L1 in MDA-MB-231 and HEK293T cells transfected with increasing amounts of UCH-L1 WT or C90S. C HCC1806 cells with UCH-L1 depletion were transfected with UCH-L1 WT or C90S, and PKM2 expression was detected. D Immunoblotting analysis of PKM2 and UCH-L1 in UCH-L1 knockdown TNBC cells treated with or without MG132. E MDA-MB-231 cells transfected with UCH-L1 WT or C90S were treated with CHX for the indicated time. F Immunoblotting analysis of PKM2 and UCH-L1 in UCH-L1 knockdown TNBC cells treated with or without CHX for the indicated time. PKM2 expression was detected by Western blot. Quantitation of PKM2 protein level based on band intensity was shown. Results shown are representative of three independent experiments.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Western Blot, Knockdown, Transfection, Expressing, Quantitation Assay

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Flag-PKM2 and HA-ubiquitin (HA-Ub) were co-expressed with Myc-UCH-L1 WT or C90S in HEK293T cells. After MG132 treatment, IP was performed with Flag antibody, followed by immunoblotting with the indicated antibodies. B HA-Ub was co-expressed with Myc-UCH-L1 WT or C90S in MDA-MB-231 cells. After MG132 treatment, IP was performed with PKM2 antibody, followed by immunoblotting with indicated antibodies. C HCC1806 cells with or without UCH-L1 depletion were transfected with HA-Ub. IP was performed with PKM2 antibody after MG132 treatment, followed by immunoblotting with indicated antibodies. D Ubiquitinated PKM2 was purified from HEK293T cells transfected with HA-Ub, and then incubated with GST-tagged wild-type UCH-L1, and UCH-L1 C90S proteins in reaction buffer. The reactions were terminated by boiling in 1X SDS sample buffer and analyzed using Western blot with anti-HA antibody. E HA-WT, K6, K11, K27, K29, K33, K48, or K63 Ub were co-transfected with Flag-PKM2 and Myc-UCH-L1 into HEK293T cells. Cell lysates were collected after MG132 treatment for 8 h and subjected to ubiquitination assay, and the ubiquitination level of PKM2 was detected by HA antibody. F Flag-PKM2 (WT or indicated KR mutants) and HA-Ub were co-expressed in 293T cells with or without UCH-L1 knockdown. IP was performed with Flag antibody after MG132 treatment for 8 h, followed by immunoblotting with anti-HA. G HCC1806 cells expressing Flag-PKM2 WT or indicated KR mutants were transfected with UCH-L1 siRNA, followed by immunoblotting with indicated antibodies. H HCC1806 cells expressing indicated KR mutants of Flag-PKM2 were transfected with UCH-L1 siRNA, and then treated with CHX for the indicated time. Cell lysates were collected and subjected to immunoblotting analysis. Quantitation of Flag-PKM2 protein level based on band intensity was shown. Results shown are representative of three independent experiments.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Purification, Incubation, Knockdown, Expressing, Quantitation Assay

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Dose–response curves of HCC1806 and HCC1806/TaxR cells. The IC50 was determined according to a dose vs. response curve by GraphPad Prism. B Immunoblotting analysis of PKM2 and UCH-L1 in HCC1806 and HCC1806/TaxR cells. C The glucose consumption, lactate and ATP production were determined and compared in HCC1806 and HCC1806/TaxR cells. D HCC1806/TaxR cells from the indicated groups were incubated with PTX for 72 h, and cell viability was measured by CCK-8 assay. E Cell apoptosis of HCC1806/TaxR cells with indicated treatment was measured by TUNEL assay. Scale bars, 150 μm. F HCC1806/TaxR cells with or without UCH-L1 depletion were incubated with 20 nM PTX for 24 h, and cell proliferation was measured by EdU assay. Scale bars, 150 μm. G Colony formation of HCC1806/TaxR cells (300 cells per well) with or without UCH-L1 depletion in the presence of DMSO or 20 nM PTX. HCC1806/TaxR cells were subcutaneously injected into 6-week-old female nude mice and subjected to PTX treatment as planned ( n = 5). Tumor sizes were measured and calculated as curves shown ( H ). The mice tumors of each group were photographed at the termination of the experiment (I). Tumor weights were measured at the end of the experiment ( J ). Characterization of HCC1806/TaxR xenograft tumors with histologic analysis by IHC staining of Ki67 and TUNEL ( K ). Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Western Blot, Incubation, CCK-8 Assay, TUNEL Assay, EdU Assay, Injection, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Boxplot of the PKM2 mRNA expression levels in TNBC and normal breast tissues from the GEPIA2 database. B Kaplan-Meier plots for the overall survival of chemotherapy-treated TNBC patients according to PKM2 mRNA expression from the Kaplan–Meier Plotter database. C The PKM2 expressions in non-responder and responder groups in breast cancer cohorts from ROC plotter server. The clinical response was evaluated based on pathological response. D Representative images of IHC of UCH-L1 and PKM2 in 40 TNBC tissues. The correlation analysis between UCH-L1 and PKM2 expression was performed using Fisher’s Exact Test. E Quantitative expression of IHC staining of PKM2 in high or low UCH-L1 groups. F Quantitative expression of IHC staining of UCH-L1 in high or low PKM2 groups. G The positive correlation between UCH-L1 and PKM2. The Pearson correlation test was used. H Mechanistic diagram of UCH-L1-catalyzed PKM2 deubiquitination in the regulation of glycolysis and chemoresistance. Depletion of UCH-L1 led to PKM2 degradation and downregulation, thereby inhibiting glycolysis and re-sensitizing TaxR-TNBC cells to chemotherapy.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Expressing, Immunohistochemistry