Journal: iScience

Article Title: Activation of limbal epithelial proliferation is partly controlled by the ACE2-LCN2 pathway

doi: 10.1016/j.isci.2024.110534

Figure Lengend Snippet:

Article Snippet: Rabbit anti-Ace2 , Proteintech , Cat:# 21115-1-AP; RRID: AB_10732845.

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, RNA Sequencing, Software

Journal: Cell Research

Article Title: Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models

doi: 10.1038/s41422-020-00450-0

Figure Lengend Snippet: a Workflow indicating identification of ACE2-binding peptides. After virtual screening, for each high-scoring candidate peptide, in vitro experiments were performed to validate its binding ability to ACE2. Picture represents 3D structure of ACE2 (PDB ID: 3D0G) and binding site (red sphere) for virtual screening. Key residue side chains are shown in blue. b Top 3D structure represents surface analysis of the binding site of ACE2 with SARS-COV-2 spike protein. Red circle marks binding site of ACE2 with SARS-COV-2 spike protein. Red and blue arrows show high charge areas in pocket by creating an interpolated charge surface; green and purple arrows point to porose area and hydrophobic domain, respectively. Middle 3D complex structure represents interaction between protein ACE2 and peptide drug dalbavancin after molecular dynamics simulation. Red structure represents dalbavancin, and green residues represent the four residues (Glu329, Gln325, Gln42, and Asp38) important for the binding of SARS-CoV-2 spike to ACE2. Lime, pale green, mint, and violet dashed lines in bottom picture represent hydrogen bonds, van der Waals bonds, Pi-Donor hydrogen bonds, and Pi-Alkyl bonds, respectively.

Article Snippet: Briefly, dalbavancin (10 μg, HY-17586, MedChemExpress), point mutated ACE2 (2 μg), anti-ACE2 antibody (1 μg, 10108-T60, Sino Biological), and protein A agarose (40 μl, 15918014, ThermoFisher) were incubated overnight at 4 °C.

Techniques: Binding Assay, In Vitro, Residue

Journal: Cell Research

Article Title: Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models

doi: 10.1038/s41422-020-00450-0

Figure Lengend Snippet: a ACE2 (0.5 μg) and SARS-CoV-2 spike protein (0.5 μg) were mixed and treated with various candidate peptide drugs (10 μM). Co-precipitated proteins were identified by western blot analysis using anti-ACE2 antibody. Analyzed proteins are indicated on the right. For positive control (bottom), ACE2-His (0.5 μg) and SARS-CoV-2 spike protein (0.5 μg) were mixed and treated with various concentration of ACE2-hFc, and then co-precipitated proteins were identified by western blot analysis using anti-His-tag antibody. b ACE2 (2 μg/ml) was crosslinked to microplates by N-oxysuccinimide esters for ELISA. SARS-CoV-2 spike protein (10 ng/mL) and tested candidate drugs (1 μM) were incubated with ACE2, and extra un-crosslinked ACE2 protein (100 ng/mL) was used as a positive control. SARS-CoV-2 spike protein antibodies were used for chromogenic reaction. c Binding curves of immobilized human ACE2 with SARS-CoV-2 spike protein (left, positive control) and dalbavancin (right). Concentration-response SPR experiment showing binding of dalbavancin to ACE2 with an equilibrium dissociation constant (K D ) of ~147 nM.

Article Snippet: Briefly, dalbavancin (10 μg, HY-17586, MedChemExpress), point mutated ACE2 (2 μg), anti-ACE2 antibody (1 μg, 10108-T60, Sino Biological), and protein A agarose (40 μl, 15918014, ThermoFisher) were incubated overnight at 4 °C.

Techniques: Western Blot, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Journal: Cell Research

Article Title: Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models

doi: 10.1038/s41422-020-00450-0

Figure Lengend Snippet: a Vero E6 cells were pre-incubated with indicated concentrations of dalbavancin for 1 h and cells were infected with SARS-CoV-2 at an MOI of 0.01. After 72 h, viral yield in cell supernatant was quantified by qRT-PCR. Cytotoxicity of these drugs to Vero E6 cells was measured by CCK-8 assays. Left and right y-axis of graphs represent mean % inhibition of virus yield and cytotoxicity of drugs, respectively. Experiments were performed in triplicate. CC 50 , half-cytotoxic concentration; SI, selectivity index. b After 1 h pre-incubation with different doses of dalbavancin, Caco-2 cells were infected with SARS-CoV-2 at an MOI of 0.01 for 72 h. The viral yield in the cell supernatant was quantified by qRT-PCR. c Inhibition of SARS-CoV-2 spike pseudo-virion entry by dalbavancin. HEK293/hACE2 cells were pre-incubated with indicated concentrations of dalbavancin, and then SARS-CoV-2 pseudo-virions were added to HEK293/hACE2 cells. At 48 hpi, transduction efficiency was measured according to luciferase activities. Means ± SD from at least three independent experiments with technical triplicates are shown. d , e Time-of-addition experiment of dalbavancin. For “Full-time” treatment, Vero E6 cells were pre-treated with testing drug (1 μM) for 0.5 h, and virus was then added to allow attachment for 1 h. Afterwards, the virus–drug mixture was removed, and the cells were cultured with dalbavancin (1 μM) at 24 hpi. For “Entry” treatment, dalbavancin (1 μM) was added to the cells for 0.5 h before viral attachment, and at 1 hpi., the virus–drug mixture was replaced with fresh culture medium and maintained for 24 h. For “Post-entry” experiment, dalbavancin (1 μM) was added at 0.5 hpi, and maintained for 24 h. Cells were infected with SARS-CoV-2 at an MOI of 0.05, and virus yield in the infected cell supernatants was quantified by qRT-PCR ( d ) and NP expression in infected cells was analyzed by western blot ( e ). f ACE2 mutant (2 μg) was incubated with dalbavancin (10 μg), anti-ACE2 antibody (1 μg), and protein A agarose (40 μL) overnight. After co-IP treatment, dalbavancin was analyzed by LC-MS/MS. Wild-type ACE2 (WT) and PBS were used as positive and negative control, respectively. Statistical significance was measured by two-way analysis of variance (ANOVA) compared with control group. ns, not significant, ** P < 0.05, ** P < 0.01.

Article Snippet: Briefly, dalbavancin (10 μg, HY-17586, MedChemExpress), point mutated ACE2 (2 μg), anti-ACE2 antibody (1 μg, 10108-T60, Sino Biological), and protein A agarose (40 μl, 15918014, ThermoFisher) were incubated overnight at 4 °C.

Techniques: Incubation, Infection, Quantitative RT-PCR, CCK-8 Assay, Inhibition, Virus, Concentration Assay, Transduction, Luciferase, Cell Culture, Expressing, Western Blot, Mutagenesis, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Negative Control, Control

Journal: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 1. DHA reduces both ACE1 and ACE2 levels in key rat tissues. Western blotting was used to measure ACE1 and ACE2 levels relative to total protein load, as

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 2. DHA differentially modulates ACE1 and ACE2 levels in growing and quiescent EA.hy926 en- dothelial cells. Western blotting was used to compare ACE1 and ACE2 relative to total protein load, as measured by Ponceau S in growing cells treated with ALA, EPA, or DHA at the indicated concentrations (µM) for (a) 8 and (b) 24 h, as well as in quiescent cells treated with n3-PUFA for (c) 8 and (d) 24 h. The band intensities of ACE2 and ACE1 were quantified and are graphically presented in panels (e,f), respectively; the 100 kDa band (grey bars) represents non-glycosylated ACE2, whereas the 130 kDa band (black bars) corresponds to N-glycosylated ACE2. Data are presented as mean ± SEM, n = 3; bars not sharing a common letter in the graphs are significantly different (p < 0.05) based on Duncan’s multiple range or LSD post-hoc tests.

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 5. Schematic of proposed mechanism of action. LCn3-PUFAs, EPA and DHA, reduce ACE2 protein levels. These n3-PUFAs may also decrease ACE2 glycosylation. These mechanisms may explain how DHA blocks SARS-CoV-2 pseudovirus entry into HEK293 cells. LCn3-PUFAs also downregulate ACE1 protein levels, thus maintaining the balance between ACE1 and ACE2, and diminishing the risk of adverse CVD outcomes. This figure was prepared by S. Huang using Microsoft PowerPoint software version 16.16.27.

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Glycoproteomics, Software

Journal: Environmental Science and Pollution Research International

Article Title: Rutin protects against gamma-irradiation and malathion-induced oxidative stress and inflammation through regulation of mir-129-3p, mir-200C-3p, and mir-210 gene expressions in rats’ kidney

doi: 10.1007/s11356-023-27166-z

Figure Lengend Snippet: The activity of acetylcholinesterase, angiotensin I converting enzyme, and angiotensin II converting enzyme in the kidney tissues. C, control; rutin, rutin-treated animals; IRR, gamma-irradiated animals; MT, malathion-treated animals; IRR/MT, gamma-irradiation/malathion-treated animals; IRR/rutin, gamma-irradiation/rutin-treated animals; MT/rutin, malathion/rutin-treated animals; IRR/MT/rutin, gamma-irradiation/malathion/rutin-treated animals; AchE, acetylcholinesterase; ACE I, angiotensin I converting enzyme. The statistical significance to control, IRR, MT, and IRR/MT are denoted by a, b, c, and d, respectively, at p < 0.05. Statistical significance was analyzed by one-way ANOVA with Tukey post hoc multiple comparisons. These enzymes are assessed by ELISA technique

Article Snippet: Elabscience ELISA commercial kits for rats were used to assess the activity of angiotensin-converting enzymes ACE I (Cat. No: E-EL-R2401) and ACE II (Cat. No: E-EL-R2453), in the rats’ kidneys tissues, according to the catalogs’ instructions.

Techniques: Activity Assay, Control, Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Physiological reports

Article Title: Inhibition of ubiquitin-specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts.

doi: 10.14814/phy2.14193

Figure Lengend Snippet: Figure 7. Effects of ML364 on cytotoxicity, proliferation, intracellular level of ATP, mitochondrial membrane potential, mitochondria morphology, and accumulation of ROS in C2C12 cells. C2C12 cells were treated with ML364 (10 µmol/L) or vehicle (DMSO) for 12 h (A), 5 days (B), 8 h (C, D), 4 h (E), or 0.5–2 h (F). (A) LDH content in the culture medium. (B) BrdU incorporation assay. Cells (500 cells) were seeded in a 96-well plate, incubated in the presence of BrdU with the inhibitor or vehicle for 5 days, and subjected to the BrdU ELISA assay. (C) Intracellular content of ATP. Values were calculated as ATP content per single cell, and are represented as relative to those detected in control cells. (D) The mitochondrial membrane potential was evaluated by staining with 20 nmol/L TMRM. The fluorescence intensity of cells was analyzed using a flow cytometer. After subtraction of the fluorescence intensity of nonstained cells, the median fluorescent signal values were calculated. (E) Morphology of mitochondria. After treatment with vehicle (left) or ML364 (right), the cells were stained with MitoTracker Red CMXRos. Scale bars represent 10 µm. (F) Mitochondrial accumulation of ROS. For the last 30 min of incubation, the cells were treated with the mitochondrial ROS indicator MitoSOX Red. Scale bars represent 20 µm. Nuclei were stained with Hoechst33342. Data are expressed as means one standard deviation of six (A-C) or seven (D) wells of a representative experiment of three experimental series.*P < 0.05 versus vehicle-treated cells (B-D). P-values obtained from Student’s t-test are also shown (A). Microscopic images are representative of three experiments (E, F).

Article Snippet: In some experiments, we treated the cells with the USP2-selective inhibitor ML364 (10 μmol/L) (MedChemExpress, Monmouth Junction, NJ, USA) or vehicle (DMSO, 2 mmol/L) for 0.5, 2, 4, 8, 12 h, or 5 days.

Techniques: Membrane, BrdU Incorporation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Single Cell, Control, Staining, Cytometry, Standard Deviation

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: The association between USP2 expression and clinicopathological parameters in bladder cancer patients.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Abnormally elevated expression of USP2 in BLCA tissues and cell lines. (A) RT-qPCR analysis of USP2 level in tumor tissues and adjacent normal tissues of 46 BLCA patients. (B) The protein level of USP2 was examined in 4 BLCA patients. (C) RT-qPCR was used to test USP2 expression in BLCA tissues. ΔCTN represented comparative Ct in normal tissues. ΔCTC was comparative Ct in tumor tissues. (D) Kaplan-Meier survival analysis of the relationship between USP2 level and overall survival of BLCA patients. (E, F) RT-qPCR and western blot analysis of USP2 level in BLCA cell lines and normal bladder epithelial cells SV-HUC-1. Values are mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Depletion of USP2 weakened cell proliferation, migration and invasion of BLCA cells. (A, B) USP2 expression was extremely decreased in 5637 and J82 cells transfected with sh-USP2#1 or sh-USP2#2. (C) CCK-8 assay displayed cell growth curves in 5637 and J82 cells. (D) Representative images and quantification of EdU assay were performed in BLCA cells. (E) The migration of BLCA cells was assessed using the wound healing assay. (F) Transwell assay was used to test the capacity of cell invasion in BLCA cells. Results expressed as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Migration, Expressing, Transfection, CCK-8 Assay, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: EZH2 was increased in BLCA and correlated with USP2 level. (A) RT-qPCR analysis of EZH2 level in tumor tissues and adjacent normal tissues of 46 BLCA patients. (B) RT-qPCR was used to test EZH2 expression in BLCA tissues. (C) Kaplan-Meier survival analysis of the relationship between EZH2 level and overall survival of BLCA patients. (D) The correlation between USP2 and EZH2 levels in BLCA tissues was assessed using Spearman analysis. (E) RT-qPCR analysis of EZH2 level in BLCA cell lines and normal bladder epithelial cells. (F) RT-qPCR detection of EZH2 expression in 5637 and J82 cells transfected with sh-USP2. (G) Western blot analysis of EZH2 and H3K27me3 levels in 5637 and J82 cells transfected with sh-USP2. Data are represented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

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

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: USP2 stabilized EZH2 by inhibiting its ubiquitination and degradation in BLCA cells. (A) 5637 and J82 cell lysates were immunoprecipitated using antibodies against USP2 or EZH2, followed by immunoblotting with the indicated antibodies. (B) BLCA cells were transfected with His-USP2 and HA-EZH2. Cell lysates were immunoprecipitated using anti-His magnetic beads, and immunoblotted with His and HA antibodies. (C) Cells overexpressing USP2 were treated with 50 ng/ml Cycloheximide (CHX) for 3, 6, and 9 h. Western blot was used to detect EZH2 expression. (D) Western blot was used to detect the expressions of USP2 and EZH2 in cells transfected with sh-NC or sh-USP2 with/without proteasomal inhibitor MG132 (20 μM) for 5 h. (E) EZH2 in BLCA cells transfected with His-USP2 or sh-USP2 was immunoprecipitated to detect ubiquitylation. Data are represented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection, Magnetic Beads, Expressing

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Overexpressed EZH2 abolished USP2 silencing-mediated biological functions on BLCA cells. We knocked down the level of USP2 and overexpressed EZH2 expression in 5637 cells. (A) The CCK-8 assay displayed cell growth curves in 5637 cells. (B) Representative images and quantification of EdU assay in 5637 cells. (C) Transwell detection of invasion in 5637 cells. (D) Western blot analysis of USP2 and EZH2 protein levels. Values were expressed as mean ± SD of three separate determinations. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, CCK-8 Assay, EdU Assay, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: USP2 epigenetically repressed SOX1 expression via EZH2 stabilization. RT-qPCR was used to detect the levels of candidate gene in BLCA cells transfected with sh-USP2 (A) or si-EZH2 (B). (C, D) ChIP assay was performed to assess the occupancy of EZH2 and H3K27me3 in the SOX1 promoter region in cells with sh-NC or sh-USP2. (E, F) RT-qPCR and western blot analysis of EZH2 and SOX1 expression in BLCA cells transfected with sh-USP2 and/or pcDNA3.1-EZH2. Error bars stand for the mean ± SD of at least triplicate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

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

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: The USP2-EZH2-SOX1 axis in tumor growth of BLCA in vivo . The 5637 cells stably expressing sh-USP2 or EZH2 were separately injected into the flanks of BALB/c nude mice to establish a xenograft tumor model. (A) Representative photographs of tumors from the xenograft nude mice. (B) Tumor weight in the three groups. (C) Tumor volumes were determined on the indicated days. (D) IHC analysis detected the expression of SOX1 in tumor tissues. (E) Schematic graph illustrating the role of USP2 in regulating the progression of BLCA. The results are representative of three independent experiments. All data are represented as mean ± SD. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: In Vivo, Stable Transfection, Expressing, Injection