Journal: Pharmacological research

Article Title: Curcumin modulates β-catenin stabilization via targeting proteasomal deubiquitinating enzyme USP14.

doi: 10.1016/j.phrs.2025.107745

Figure Lengend Snippet: Figure 3. Curcumin directly binds to USP14 in proteasome.

Article Snippet: For purified USP14 protein (HY-P71418, MedChemExpress), 40 nM USP14 protein were incubated with 20/50/100 μM curcumin for 1 h at room temperature, followed incubated with 1 μM HA-UbVS (U-212-025, R&D systems) for 30 min at 37°C and analyzed using Western blot.

Techniques:

Journal: Pharmacological research

Article Title: Curcumin modulates β-catenin stabilization via targeting proteasomal deubiquitinating enzyme USP14.

doi: 10.1016/j.phrs.2025.107745

Figure Lengend Snippet: Figure 4. USP14 is required for curcumin stabilization phospho-β-catenin.

Article Snippet: For purified USP14 protein (HY-P71418, MedChemExpress), 40 nM USP14 protein were incubated with 20/50/100 μM curcumin for 1 h at room temperature, followed incubated with 1 μM HA-UbVS (U-212-025, R&D systems) for 30 min at 37°C and analyzed using Western blot.

Techniques:

Journal: Pharmacological research

Article Title: Curcumin modulates β-catenin stabilization via targeting proteasomal deubiquitinating enzyme USP14.

doi: 10.1016/j.phrs.2025.107745

Figure Lengend Snippet: Figure 5. Curcumin enhances USP14-mediated PBC trapping and modulates proteasome associations.

Article Snippet: For purified USP14 protein (HY-P71418, MedChemExpress), 40 nM USP14 protein were incubated with 20/50/100 μM curcumin for 1 h at room temperature, followed incubated with 1 μM HA-UbVS (U-212-025, R&D systems) for 30 min at 37°C and analyzed using Western blot.

Techniques:

Journal: Pharmacological research

Article Title: Curcumin modulates β-catenin stabilization via targeting proteasomal deubiquitinating enzyme USP14.

doi: 10.1016/j.phrs.2025.107745

Figure Lengend Snippet: Figure 6. Modulation of USP14 and PBC by curcumin leads to mitotic abnormalities and cancer inhibition.

Article Snippet: For purified USP14 protein (HY-P71418, MedChemExpress), 40 nM USP14 protein were incubated with 20/50/100 μM curcumin for 1 h at room temperature, followed incubated with 1 μM HA-UbVS (U-212-025, R&D systems) for 30 min at 37°C and analyzed using Western blot.

Techniques: Inhibition

Journal: Frontiers in Molecular Biosciences

Article Title: A prognostic nomogram for colorectal cancer based on ubiquitin-specific protease 21 expression: a retrospective cohort study

doi: 10.3389/fmolb.2026.1764916

Figure Lengend Snippet: Expression and clinical significance of USP21 in CRC based on public database analysis. (A) USP21 transcriptional expression levels across various malignant tumor tissues based on TIMER database. (B) USP21 expression levels in CRC from TCGA database. (C) USP21 expression levels in the GSE83889 dataset. (D) ROC curve for USP21 expression in diagnosing TCGA-CRC. (E) Kaplan-Meier survival curves for overall survival (OS) stratified by USP21 expression in TCGA-CRC.

Article Snippet: USP21 protein expression was assessed by streptavidin–peroxidase immunohistochemistry using a rabbit monoclonal anti-USP21 antibody (1:400, Boster, A06639-1).

Techniques: Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: A prognostic nomogram for colorectal cancer based on ubiquitin-specific protease 21 expression: a retrospective cohort study

doi: 10.3389/fmolb.2026.1764916

Figure Lengend Snippet: Upregulation of USP21 correlates with poor prognosis in CRC patients. (A) Representative immunohistochemical staining of USP21 in CRC tissues. (B) Kaplan–Meier survival curves for high and low USP21 expression groups.

Article Snippet: USP21 protein expression was assessed by streptavidin–peroxidase immunohistochemistry using a rabbit monoclonal anti-USP21 antibody (1:400, Boster, A06639-1).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Journal of Immunological Methods

Article Title: The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies

doi: 10.1016/j.jim.2022.113244

Figure Lengend Snippet: The dose responsiveness and specificity of S1RBD-ACE2 and ACE2-S1RBD binding assay. (A) Schematic diagram of the S1RBD-ACE2 binding assay. (B) Detection of ACE2- S1RBD binding interaction where ACE2 protein was coated on a microplate and S1RBD concentration was varied. (C) Schematic diagram of the ACE2-S1RBD binding assay. (D) Detection of S1RBD-ACE2 binding interaction where S1RBD protein was coated on a microplate and ACE2 concentration was varied. (E) Selectivity of binding assay for non-SARS viruses in patient sera. Data points represent individual patients seropositive for the virus indicated. Binding inhibition is the percentage of signal (OD450) in the presence of seropositive sera relative to no sera. In B and D, the average of three independent experiments is shown. Error bars indicate standard error of the mean (SEM).

Article Snippet: After being washed 3 times with PBS with 0.05% Tween 20 (PBS-T), plates were blocked with 100 μl/well of BSA blocking buffer (Rockland Inc.) After another wash, either ACE2 protein or S1RBD protein, depending on the coated antigen, was added at 100 μl/well in a range of concentrations from 100 to 0 ng/mL to the coated plate and incubated at room temperature (RT) for 2.5 h. Plates were washed with PBS-T, and 100 μl/well of a 1:1,00 dilution of HRP-conjugated IgG antibody was added and incubated for 1 h at RT.

Techniques: Binding Assay, Concentration Assay, Virus, Inhibition

Journal: Journal of Immunological Methods

Article Title: The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies

doi: 10.1016/j.jim.2022.113244

Figure Lengend Snippet: Neutralization antibodies and drugs screening using S1RBD-ACE2 binding assay and pseudovirus harboring the SARS-CoV-2 S protein. (A) Comparison of the consistency of the results of three different neutralizing antibodies and one antibody against Nucleocapsid (N) protein as negative control using binding assay and pseudovirus assay. All antibodies were raised in mice and clone numbers are indicated for each. (B) Binding assay and pseudovirus in the presence of five small molecules (0.4 μg/ml) with known ability to interfere with the S1RBD-ACE2 interaction as well as two that target unrelated proteins (see also ). (C) Titration of neutralizing antibodies in binding assay. (D) Titration of neutralizing antibodies in pseudovirus assay. Inhibition was calculated as percentage of signal (OD 450 or RLU for binding assay and pseudovirus assay, respectively) in the presence of inhibitor (antibody or small molecule) relative to no inhibitor. The average of 3 independent experiments was performed with error bars indicating SEM.

Article Snippet: After being washed 3 times with PBS with 0.05% Tween 20 (PBS-T), plates were blocked with 100 μl/well of BSA blocking buffer (Rockland Inc.) After another wash, either ACE2 protein or S1RBD protein, depending on the coated antigen, was added at 100 μl/well in a range of concentrations from 100 to 0 ng/mL to the coated plate and incubated at room temperature (RT) for 2.5 h. Plates were washed with PBS-T, and 100 μl/well of a 1:1,00 dilution of HRP-conjugated IgG antibody was added and incubated for 1 h at RT.

Techniques: Neutralization, Binding Assay, Comparison, Negative Control, Titration, Inhibition

Journal: Journal of Immunological Methods

Article Title: The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies

doi: 10.1016/j.jim.2022.113244

Figure Lengend Snippet: Target Molecules of the Small Molecules for Spike-RBD-ACE2 Binding Assay based on Computational Screening.

Article Snippet: After being washed 3 times with PBS with 0.05% Tween 20 (PBS-T), plates were blocked with 100 μl/well of BSA blocking buffer (Rockland Inc.) After another wash, either ACE2 protein or S1RBD protein, depending on the coated antigen, was added at 100 μl/well in a range of concentrations from 100 to 0 ng/mL to the coated plate and incubated at room temperature (RT) for 2.5 h. Plates were washed with PBS-T, and 100 μl/well of a 1:1,00 dilution of HRP-conjugated IgG antibody was added and incubated for 1 h at RT.

Techniques: Binding Assay

Journal: Journal of Immunological Methods

Article Title: The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies

doi: 10.1016/j.jim.2022.113244

Figure Lengend Snippet: Evaluation of S1RBD-ACE2 binding assay and pseudovirus assay using COVID-19 patient sera. (A) Study subjects used for validation of binding assay. (B) ROC curve generated for S1RBD-ACE2 binding assay of each patient serum sample. (C) ROC curve generated for pseudovirus inhibition test in determining SARS-CoV-2 neutralization activity. (D) Correlation between binding assay and pseudovirus assay using COVID-19 (+ve) and COVID-19 (−ve) serum samples. (E) Correlation between binding assay and pseudovirus assay using COVID-19 (+ve) serum samples alone. Inhibition was calculated as the fraction of signal (OD 450 or RLU for binding assay and pseudovirus assay, respectively) in the presence of serum relative to no serum.

Article Snippet: After being washed 3 times with PBS with 0.05% Tween 20 (PBS-T), plates were blocked with 100 μl/well of BSA blocking buffer (Rockland Inc.) After another wash, either ACE2 protein or S1RBD protein, depending on the coated antigen, was added at 100 μl/well in a range of concentrations from 100 to 0 ng/mL to the coated plate and incubated at room temperature (RT) for 2.5 h. Plates were washed with PBS-T, and 100 μl/well of a 1:1,00 dilution of HRP-conjugated IgG antibody was added and incubated for 1 h at RT.

Techniques: Binding Assay, Biomarker Discovery, Generated, Inhibition, Neutralization, Activity Assay

Journal: Journal of Immunological Methods

Article Title: The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies

doi: 10.1016/j.jim.2022.113244

Figure Lengend Snippet: Evaluation of vaccine efficacy using S1RBD-ACE2 binding assay and pseudovirus assay. (A) ACE2 binding inhibition (%) and (B) pseudovirus neutralization (%) in subjects pre- and post-vaccination. (C) Correlation study between binding assay and pseudovirus assay in post vaccinated sample. Whiskers represent min to max values and all data points are shown.

Article Snippet: After being washed 3 times with PBS with 0.05% Tween 20 (PBS-T), plates were blocked with 100 μl/well of BSA blocking buffer (Rockland Inc.) After another wash, either ACE2 protein or S1RBD protein, depending on the coated antigen, was added at 100 μl/well in a range of concentrations from 100 to 0 ng/mL to the coated plate and incubated at room temperature (RT) for 2.5 h. Plates were washed with PBS-T, and 100 μl/well of a 1:1,00 dilution of HRP-conjugated IgG antibody was added and incubated for 1 h at RT.

Techniques: Binding Assay, Inhibition, Neutralization

Journal: Nature Communications

Article Title: Design of quinoline SARS-CoV-2 papain-like protease inhibitors as oral antiviral drug candidates

doi: 10.1038/s41467-025-56902-x

Figure Lengend Snippet: a Differential scanning fluorimetry assay of Jun13296 in stabilizing SARS-CoV-2 PL pro . Jun12682 was included as a positive control for comparison. Data from Jun12682 is the mean of two repeats, and data from Jun13296 is the mean ± standard deviation of three technical repeats. b K i plot of Jun13296 in inhibiting SARS-CoV-2 PL pro hydrolysis of ISG15-AMC. c K i plot of Jun13296 in inhibiting SARS-CoV-2 PL pro hydrolysis of Ub-AMC. d Counter screening of Jun 13296 against host proteases USP2, USP7, USP8, USP14, USP15, USP30, UCH-L1, cathepsin B, cathepsin K, calpain-1, trypsin, and caspase 3. Data in ( d ) are presented as mean ± standard deviation of two technical repeats. Source data are provided as a file.

Article Snippet: E-520-025), 500 nM USP14 (ProSci, 91-171), 1 nM USP15 (R&D systems, E594), 20 nM USP30 (R&D systems, E582), and 1 nM UCH-L1 (R&D systems, 6007-CY).

Techniques: Fluorimetry Assay, Positive Control, Comparison, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: The Location of Asparagine-linked Glycans on West Nile Virions Controls Their Interactions with CD209 (Dendritic Cell-specific ICAM-3 Grabbing Nonintegrin)

doi: 10.1074/jbc.m605429200

Figure Lengend Snippet: FIGURE 1. Flavivirus RVPs containing N-linked glycans at E protein residue 67 infect CD209-expressing cells efficiently. A, Western blot analysis of prM and M content of RVP stocks made by transfection of replicon- containing cells with plasmids encoding the WNV structural proteins, plus pcDNA3 () or pcDNA3.1furin (). B–F, RVPs were made with expression plasmids for flavivirus structural proteins and human furin as in A. Serial dilutions of RVPs were used to infect K562 control cells (closed black diamonds), K562-CD209 cells (open red squares), or K562-CD209L cells (closed blue triangles). Renilla luciferase activity was measured 48 h after infec- tion.B,RVPsweremadewithexpressionplasmidsforDENVcapsidandDENVserotype1(Westpacstrain)prM-E. C–F, RVPs were made with plasmids encoding WNV capsid and wild-type or glycosylation mutant WNV prM-E constructs. Numbers above B–F indicate the locations of N-linked glycosylation sites within the E proteins used. Similar results were seen in more than four separate experiments using independent RVP preparations. The horizontal line on each graph represents the average background luciferase activity for uninfected wells.

Article Snippet: Polyclonal rabbit antibodies against prM were generated by ProSci (San Diego) by immunization of rabbits with peptides spanning the following three regions in prM: the N-terminal “pr” region, the furin cleavage junction, and the C-terminal “M” region.

Techniques: Residue, Expressing, Western Blot, Transfection, Control, Luciferase, Activity Assay, Glycoproteomics, Mutagenesis, Construct

Journal: Clinical Science

Article Title: Antiepileptic effects of long-term intracerebroventricular infusion of angiotensin-(1-7) in an animal model of temporal lobe epilepsy

doi: 10.1042/cs20200514

Figure Lengend Snippet: Figure 7. Effects of TLE and Ang-(1-7) treatment on hippocampal protein levels Representative Western blot bands (A) and protein levels of ACE2 (B), NEP (C), AT2R (D), AT1R (*P=0.0406 CT vs EP) (E), Mas [*P=0.0406 CT vs EP + Ang-(1-7)] (F), IL-6 (G), SOD (H), CAT (*P=0.0389 CT vs EP) (I), Bcl-2 (*P=0.0468 CT vs EP) (J), and

Article Snippet: Published by Portland Press Limited on behalf of the Biochemical Society 2265 D ow nloaded from http://portlandpress.com /clinsci/article-pdf/134/17/2263/892258/cs-2020-0514.pdf by U niversidade Federal de Sao Paulo (U N IFESP) user on 14 M ay 2024 Table 1 Host, specificity, dilution, catalog number and source of the antibodies used in the present study Antibody Host Specificity Dilution Catalog number Source ACE2 Rabbit Monoclonal 1:1000 GTX01160 GeneTex, CA, U.S.A. NEP Mouse Monoclonal 1:1000 sc-46656 Santa Cruz Biotechnology, CA, U.S.A. AT1 Mouse Monoclonal 1:500 sc-515884 Santa Cruz Biotechnology, CA, U.S.A. AT2 Rabbit Monoclonal 1:1000 M00432 Boster Biological Technology, CA, U.S.A. Mas Rabbit Polyclonal 1:500 ab66030 Abcam, MA, U.S.A. IL-6 Mouse Monoclonal 1:500 IM-0407 Imuny Biotechnology, SP, Brazil SOD Mouse Monoclonal 1:1000 sc-17767 Santa Cruz Biotechnology, CA, U.S.A. CAT Mouse Monoclonal 1:1000 LS-B2554 Lifespan Biosciences, WA, U.S.A. Bcl-2 Mouse Monoclonal 1:1000 sc-7382 Santa Cruz Biotechnology, CA, U.S.A. mTOR Mouse Monoclonal 1:1000 #4517 Cell Signaling Technology, MA, U.S.A. Phospho-mTOR Rabbit Polyclonal 1:1000 #2971 Cell Signaling Technology, MA, U.S.A. GAPDH Mouse Monoclonal 1:2000 sc-365062 Santa Cruz Biotechnology, CA, U.S.A. 2266 © 2020 The Author(s).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The steroid hormone 20-hydroxyecdysone induces lipophagy via the brain-adipose tissue axis by promoting the adipokinetic hormone pathway

doi: 10.1016/j.jbc.2025.108179

Figure Lengend Snippet: Knockdown of Atg8 and Orp8 blocked lipophagy. A , interference efficiency was detected by Western blotting. B , the colocalization of lysosomes and LDs in fat body cells after dsAtg8 injection. Lysosomes were stained with Lyso-Tracker ( red ), and LDs were stained with BODIPY ( green ). Yellow indicates examples of Lyso-Tracker-positive structures containing LDs. The ruler represents 50 μm. Bi , statistical analysis of autolysosome numbers in ( B ). C , the levels of triglycerides in the fat body after Atg8 knockdown. D , the location of ORP8 in fat body cells. Red fluorescence represents ORP8; green fluorescence represents LDs. The scale bar is 50 μm. E , the specificity of the ORP8 antibody in the fat body was analyzed by Western blotting. F , the expression of ORP8 was analyzed by Western blotting. G and Gi , the colocalization of LDs and lysosomes was detected after Orp8 knockdown. The ruler represents 50 μm. H , the levels of triglycerides in the fat body after Orp8 knockdown. I , phenotypes after dsOrp8 injection. Bars represent 1 cm. dsGfp was used as a control. J , statistical analysis of the phenotypes in ( I ). The pupation time was sixth instar 0 h larvae to pupae. K , the average weight of pupae and pupal phenotypes. L , percentage of different phenotypes from pupae to adults. The bars indicate the mean ± SD. p values and asterisks indicate differences by two-tailed Student's t test (∗ p < 0.05, ∗∗ p < 0.01). The different lowercase letters indicate significant differences in ANOVA ( p < 0.05). LD, lipid droplet; ORP8, oxysterol-binding protein-related protein 8.

Article Snippet: Polyclonal anti-ORP8 antibody (anti-OSBPL8 antibody) was obtained from Boster Biological Technology.

Techniques: Knockdown, Western Blot, Injection, Staining, Fluorescence, Expressing, Control, Two Tailed Test, Binding Assay