Journal: EMBO Molecular Medicine

Article Title: Targeting USP11 regulation by a novel lithium-organic coordination compound improves neuropathologies and cognitive functions in Alzheimer transgenic mice

doi: 10.1038/s44321-024-00146-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: USP11 Recombinant Protein, Human (Sf9) , MedChemExpress , Cat:#HY-P702063.

Techniques: Recombinant, Sequencing, Software, Real-time Polymerase Chain Reaction, Microscopy, Enzyme-linked Immunosorbent Assay, Staining, CCK-8 Assay, Activity Assay, BIA-KA, Reverse Transcription

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: Biology direct

Article Title: USP44 regulates HEXIM1 stability to inhibit tumorigenesis and metastasis of oral squamous cell carcinoma.

doi: 10.1186/s13062-024-00573-z

Figure Lengend Snippet: Fig. 1 Downregulation of USP44 in OSCC. (A) Volcano plot: all differentially expressed coding mRNAs from the GSE37991 (https://www.ncbi.nlm.nih.gov/ geo) and The Cancer Genome Atlas (TCGA, https://www.cancer.gov/ccg/research/genome-sequencing/tcga) databases. (B) Venn diagrams: differentially expressed genes (DEGs) identified from GSE37991 and TCGA data sets. The overlaps display the up- or down-regulated DEGs common to both datasets. (C) Kaplan-Meier survival analysis of OSCC patients in TCGA data sets. (D and E) The expression levels of USP44 in OSCC tumor tissues and adjacent normal tissues using data from GSE37991, TCGA and samples from The First Affiliated Hospital of Zhengzhou University, respectively. (F) Representative immuno histochemical staining of USP44 in human OSCC tissues

Article Snippet: After blocking in 1% BSA for 15 min, the tissue samples were incubated with primary antibodies USP44 (1:50, A08401-2, Boster, Wuhan, China), CD133 (1:50, 18470-1-AP, Proteintech) and Ki67 (1:50, AF0198, Affbiotech, Changzhou, China) at 4 ̊C overnight, followed by secondary antibody HRP-conjugated Goat Anti-Rabbit IgG (1:500, #31460, ThermoFisher Scientific, Pittsburgh, PA, USA) for 60 min at 37 ̊C.

Techniques: Sequencing, Expressing, Staining

Journal: Biology direct

Article Title: USP44 regulates HEXIM1 stability to inhibit tumorigenesis and metastasis of oral squamous cell carcinoma.

doi: 10.1186/s13062-024-00573-z

Figure Lengend Snippet: Fig. 2 USP44 inhibits OSCC cell proliferation, migration, and invasion in vitro. (A-B) CCK-8 assays showing that cell viability was enhanced in USP44- silenced OSCC cells but was decreased in USP44-overexpressed OSCC cells. (C-D) Clonogenic assays depicting the effects of USP44 on OSCC cell prolifera tion. (E-F) Transwell assays analyzing the migration and invasion capabilities of OSCC cells with altered USP44 expression. (G-H) Quantitation of Transwell assays in E and F

Article Snippet: After blocking in 1% BSA for 15 min, the tissue samples were incubated with primary antibodies USP44 (1:50, A08401-2, Boster, Wuhan, China), CD133 (1:50, 18470-1-AP, Proteintech) and Ki67 (1:50, AF0198, Affbiotech, Changzhou, China) at 4 ̊C overnight, followed by secondary antibody HRP-conjugated Goat Anti-Rabbit IgG (1:500, #31460, ThermoFisher Scientific, Pittsburgh, PA, USA) for 60 min at 37 ̊C.

Techniques: Migration, In Vitro, CCK-8 Assay, Expressing, Quantitation Assay

Journal: Biology direct

Article Title: USP44 regulates HEXIM1 stability to inhibit tumorigenesis and metastasis of oral squamous cell carcinoma.

doi: 10.1186/s13062-024-00573-z

Figure Lengend Snippet: Fig. 3 USP44 inhibits stemness of OSCC cells. (A-B) Representative microscopy images of monolayer and spherical OSCC cells. The mRNA levels of stem cell markers CD133, OCT4, SOX2 and Nanog were detected by RT-qPCR. (C-D) The expression of USP44 in monolayer and spherical OSCC cells, respec tively, using RT-qPCR and western blotting assays. (E) Representative microscopy images of spheroid formation in OSCC cells. (F) RT-qPCR quantification of CD133, OCT4, SOX2 and Nanog mRNA levels in the indicated OSCC cells. mc, monolayer cell; sc, spherical cell

Article Snippet: After blocking in 1% BSA for 15 min, the tissue samples were incubated with primary antibodies USP44 (1:50, A08401-2, Boster, Wuhan, China), CD133 (1:50, 18470-1-AP, Proteintech) and Ki67 (1:50, AF0198, Affbiotech, Changzhou, China) at 4 ̊C overnight, followed by secondary antibody HRP-conjugated Goat Anti-Rabbit IgG (1:500, #31460, ThermoFisher Scientific, Pittsburgh, PA, USA) for 60 min at 37 ̊C.

Techniques: Microscopy, Quantitative RT-PCR, Expressing, Western Blot

Journal: Biology direct

Article Title: USP44 regulates HEXIM1 stability to inhibit tumorigenesis and metastasis of oral squamous cell carcinoma.

doi: 10.1186/s13062-024-00573-z

Figure Lengend Snippet: Fig. 4 USP44 suppresses tumorigenic abilities of OSCC cells in vivo. (A-B) Images of subcutaneous tumors from mice. The tumor growth curves (volume) over the course of the study were also shown. (C-D) Representative immunohistochemical staining images of tumor tissues for USP44, CD133, and Ki67

Article Snippet: After blocking in 1% BSA for 15 min, the tissue samples were incubated with primary antibodies USP44 (1:50, A08401-2, Boster, Wuhan, China), CD133 (1:50, 18470-1-AP, Proteintech) and Ki67 (1:50, AF0198, Affbiotech, Changzhou, China) at 4 ̊C overnight, followed by secondary antibody HRP-conjugated Goat Anti-Rabbit IgG (1:500, #31460, ThermoFisher Scientific, Pittsburgh, PA, USA) for 60 min at 37 ̊C.

Techniques: In Vivo, Immunohistochemical staining, Staining

Journal: Biology direct

Article Title: USP44 regulates HEXIM1 stability to inhibit tumorigenesis and metastasis of oral squamous cell carcinoma.

doi: 10.1186/s13062-024-00573-z

Figure Lengend Snippet: Fig. 5 USP44 reduces lung metastasis of OSCC cells in vivo. (A) Images of lung tissues from mice. (B) Representative H&E staining of lung tissues. (C-D) Quantification of pulmonary nodules in histograms

Article Snippet: After blocking in 1% BSA for 15 min, the tissue samples were incubated with primary antibodies USP44 (1:50, A08401-2, Boster, Wuhan, China), CD133 (1:50, 18470-1-AP, Proteintech) and Ki67 (1:50, AF0198, Affbiotech, Changzhou, China) at 4 ̊C overnight, followed by secondary antibody HRP-conjugated Goat Anti-Rabbit IgG (1:500, #31460, ThermoFisher Scientific, Pittsburgh, PA, USA) for 60 min at 37 ̊C.

Techniques: In Vivo, Staining

Journal: Biology direct

Article Title: USP44 regulates HEXIM1 stability to inhibit tumorigenesis and metastasis of oral squamous cell carcinoma.

doi: 10.1186/s13062-024-00573-z

Figure Lengend Snippet: Fig. 7 USP44 enhances the stability of HEXIM protein. (A) Western blotting showing the expression of HEXIM in CAL-27 and SCC-9 cells. (B) Co-IP with anti-USP44 or anti-HEXIM1 antibody in CAL-27 and SCC-9 cells showing the endogenous interaction between USP44 and HEXIM1. (C) Co-IP with anti-Flag or anti-Myc antibody showing the interaction between Flag-USP44 and Myc-HEXIM1 overexpressed in 293T cells. (D) Effect of overexpression of USP44 on HEXIM1 stability. CAL-27 cells were transfected with either empty vector or USP44-expressing vector and then treated with CHX for the indicated periods of time. (E) Effects of USP44 overexpression on HEXIM1 ubiquitination. 293T cells transfected with Flag-USP44, Myc-HEXIM1 or HA-ubiquitin (Ub) plasmids were subjected to IP with anti-Myc antibody and then immunoblotted with the anti-HA or anti-Myc antibody

Article Snippet: After blocking in 1% BSA for 15 min, the tissue samples were incubated with primary antibodies USP44 (1:50, A08401-2, Boster, Wuhan, China), CD133 (1:50, 18470-1-AP, Proteintech) and Ki67 (1:50, AF0198, Affbiotech, Changzhou, China) at 4 ̊C overnight, followed by secondary antibody HRP-conjugated Goat Anti-Rabbit IgG (1:500, #31460, ThermoFisher Scientific, Pittsburgh, PA, USA) for 60 min at 37 ̊C.

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, Over Expression, Transfection, Plasmid Preparation, Ubiquitin Proteomics

Journal: Biology direct

Article Title: USP44 regulates HEXIM1 stability to inhibit tumorigenesis and metastasis of oral squamous cell carcinoma.

doi: 10.1186/s13062-024-00573-z

Figure Lengend Snippet: Fig. 8 HEXIM1 mediates the antitumor effects of USP44 in OSCC cells. (A) Expression of HEXIM1 in CAL-27 cells transfected with the indicated shRNA plasmids and detected using Western blotting and RT-qPCR, respectively. (B) CCK-8 assays showing that cell viability was enhanced in HEXIM1-silenced CAL-27 cells. (C) Transwell assays analyzing the migration and invasion capabilities of CAL-27 cells with HEXIM1 silence. Quantification of migration and invasion cells was shown in the histograms. (D) Effect of HEXIM1 silencing on cell viability in CAL-27 cells overexpressing USP44. Cells were transfected with the indicated plasmids and the viability was determined by CCK-8 assay. (E) Effect of HEXIM1 silencing on cell migration and invasion capabilities in CAL-27 cells overexpressing USP44. Cells were transfected with the indicated plasmids. The migration and invasion capabilities were determined using Transwell assays. Quantification of migration and invasion cells was shown in the histograms

Article Snippet: After blocking in 1% BSA for 15 min, the tissue samples were incubated with primary antibodies USP44 (1:50, A08401-2, Boster, Wuhan, China), CD133 (1:50, 18470-1-AP, Proteintech) and Ki67 (1:50, AF0198, Affbiotech, Changzhou, China) at 4 ̊C overnight, followed by secondary antibody HRP-conjugated Goat Anti-Rabbit IgG (1:500, #31460, ThermoFisher Scientific, Pittsburgh, PA, USA) for 60 min at 37 ̊C.

Techniques: Expressing, Transfection, shRNA, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Migration

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