Journal: Journal of mass spectrometry : JMS

Article Title: Top-down analysis of novel synthetic branched proteins

doi: 10.1002/jms.4303

Figure Lengend Snippet: Assembly of Rub1-Rub1 homodimers monitored by Coomassie stained SDS-PAGE. A, Formation of Rub1K4F,T72R-Rub1K4F,T72R homodimer catalyzed by ubiquitin E1 and UBE2K (as E2). B, Formation of Rub1T72R-Rub1T72R homodimers catalyzed by ubiquitin E1 and UBE2S (as E2). The products are shown in two lanes: one collected after the reaction and the other after centrifugation to remove precipitated E1 and E2

Article Snippet: Enzymatic assembly of Rub1 T72R -Rub1 T72R homodimer Rub1 T72R (10 mg) was incubated with 20-μM ubiquitin-conjugating E2 enzyme UBE2S (UBE2S-UBD, Addgene #66713), 500-nM UBE1, 10 mM creatine phosphate, 5 mM MgCl 2 , 5 mM ATP, and creatine phosphokinase in 50-mM Tris-HCl buffer (pH 8) for approximately 16 hours.

Techniques: Staining, SDS Page, Ubiquitin Proteomics, Centrifugation

Journal: Aging (Albany NY)

Article Title: Ube2s-stabilized β-catenin protects against myocardial ischemia/reperfusion injury by activating HIF-1α signaling

doi: 10.18632/aging.102960

Figure Lengend Snippet: Ube2s expression is upregulated after MI/R injury. ( A ) qRT-PCR analysis of the mRNA level of Ube2s in the heart from C57BL/6 mice following 12 h, 24 h and 48 h of MI/R injury. Samples from mice receiving sham surgery were used as controls. Each group includes 8 mice. The results were normalized to β-Actin and expressed as relative to sham group. Data are mean ± SD. Data were compared with sham group using one-way ANOVA analysis. **, P < 0.01. ( B ) Western blotting analysis of the protein level of Ube2s in the heart as described in ( A ). β-Actin was used as a loading control. The representative band images are presented (left). The analysis of the relative band intensity is also presented (right). Data are mean ± SD. Data were compared with sham group using one-way ANOVA analysis. **, P < 0.01. ( C ) Immunohistochemistry analysis of Ube2s expression in the heart from mice subjected to sham or MI/R injury for 24 h as descried in ( A ). The representative images are shown (left). The analysis of percentage of positive stained cells is also depicted (right). Scale bar, 50 μm. Data are mean ± SD. Data were compared with sham group using Student’s t -test. **, P < 0.01.

Article Snippet: After the block with 5% BSA, membranes were incubated overnight at 4 °C with primary antibodies against the following targets: Ube2s (Novus Biologicals, 1:500), HIF-1α (Invitrogen, 1:2000), Bax (Abcam, 1:1000), Bcl-2 (Abcam, 1:1000), β-Catenin (Cell Signaling, 1:500) and β-Actin (Santa Cruz, 1:5000).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemistry, Staining

Journal: Aging (Albany NY)

Article Title: Ube2s-stabilized β-catenin protects against myocardial ischemia/reperfusion injury by activating HIF-1α signaling

doi: 10.18632/aging.102960

Figure Lengend Snippet: Ube2s acts to protect against MI/R injury. ( A – B ) C57BL/6 mice were intra-myocardially transfected with control siRNA (siCtrl) or Ube2s siRNA (siUbe2s) 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of Ube2s in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images ( A ) and relative band intensity analysis ( B ) are presented. Data are mean ± SD. Data were compared with sham group using Student’s t -test. **, P < 0.01; NS, not significant. ( C ) Heart samples were harvested as described in ( A ), and the mid-myocardial cross sections were prepared. The infarct size in the heart sections was quantified, and the results of percentage of size are shown. AAR/LVA, ratio of area at risk (AAR) to left ventricular area (LVA); IA/AAR, ratio of infarct area (IA) to AAR. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01; NS, not significant. ( D ) C57BL/6 mice were treated as in ( A ). The samples of serum were collected and the level of creatine phosphokinase (CPK) was quantified. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01. ( E , F ) C57BL/6 mice were intra-myocardially infected with lentivirus expressing vector control or Ube2s 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of Ube2s in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images ( E ) and relative band intensity analysis ( F ) are presented. Data are mean ± SD. Data were compared using Student’s t-test. **, P < 0.01. ( G ) Heart samples were harvested as described in ( E ), and the mid-myocardial cross sections were prepared. The infarct size in the heart sections was quantified, and the results of percentage of size are shown. AAR/LVA, ratio of area at risk (AAR) to left ventricular area (LVA); IA/AAR, ratio of infarct area (IA) to AAR. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01; NS, not significant. ( H ) C57BL/6 mice were treated as in ( E ). The samples of serum were collected and the level of creatine phosphokinase (CPK) was quantified. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01.

Article Snippet: After the block with 5% BSA, membranes were incubated overnight at 4 °C with primary antibodies against the following targets: Ube2s (Novus Biologicals, 1:500), HIF-1α (Invitrogen, 1:2000), Bax (Abcam, 1:1000), Bcl-2 (Abcam, 1:1000), β-Catenin (Cell Signaling, 1:500) and β-Actin (Santa Cruz, 1:5000).

Techniques: Transfection, Control, Western Blot, Infection, Expressing, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Ube2s-stabilized β-catenin protects against myocardial ischemia/reperfusion injury by activating HIF-1α signaling

doi: 10.18632/aging.102960

Figure Lengend Snippet: Ube2s augments HIF-1α activation and decreases apoptosis after MI/R injury. ( A – C ) C57BL/6 mice were intra-myocardially infected with lentivirus expressing vector control or Ube2s 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of HIF-1α, Bax and Bcl-2 in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images ( A ) and relative band intensity analysis of HIF-1α ( B ) and ratio of Bax/Bcl-2 ( C ) are presented. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01. ( D ) Heart samples were harvested as described in ( A ), and heart sections were prepared. The apoptosis was detected using TUNEL staining. The statistical analysis of percentage of TUNEL positive cells is shown. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01. ( E ) Heart samples were harvested as described in ( A ). The supernatants of the homogenized heart samples were collected, and the caspase-3 activity was determined. The results are expressed as the nmol AFC/h/mg protein. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01. ( F ) C57BL/6 mice were intra-myocardially transfected with control siRNA (siCtrl) or Ube2s siRNA (siUbe2s) 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of HIF-1α, Bax and Bcl-2 in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images are presented.

Article Snippet: After the block with 5% BSA, membranes were incubated overnight at 4 °C with primary antibodies against the following targets: Ube2s (Novus Biologicals, 1:500), HIF-1α (Invitrogen, 1:2000), Bax (Abcam, 1:1000), Bcl-2 (Abcam, 1:1000), β-Catenin (Cell Signaling, 1:500) and β-Actin (Santa Cruz, 1:5000).

Techniques: Activation Assay, Infection, Expressing, Plasmid Preparation, Control, Western Blot, TUNEL Assay, Staining, Activity Assay, Transfection

Journal: Aging (Albany NY)

Article Title: Ube2s-stabilized β-catenin protects against myocardial ischemia/reperfusion injury by activating HIF-1α signaling

doi: 10.18632/aging.102960

Figure Lengend Snippet: HIF-1α activation mediates Ube2s role in cardiomyocyte apoptosis and MI/R injury. ( A – C ) C57BL/6 mice were intra-myocardially infected with lentivirus expressing vector control or Ube2s in the presence or absence of transfection of control siRNA (siCtrl) or HIF-1α siRNA (siHIF-1α) 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of Ube2s, HIF-1α, Bax and Bcl-2 in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images ( A ) and relative band intensity analysis of HIF-1α and ratio of Bax/Bcl-2 ( B ) are presented. ( C ) The supernatants of the homogenized heart samples were collected, and the caspase-3 activity was determined. The results are expressed as the nmol AFC/h/mg protein. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01; NS, not significant. ( D ) Heart samples were harvested as described in ( A ), and the mid-myocardial cross sections were prepared. The infarct size in the heart sections was quantified, and the results of percentage of size are shown. AAR/LVA, ratio of area at risk (AAR) to left ventricular area (LVA); IA/AAR, ratio of infarct area (IA) to AAR. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01; *, P < 0.05; NS, not significant. ( E ) C57BL/6 mice were treated as in ( A ). The samples of serum were collected and the level of creatine phosphokinase (CPK) was quantified. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01.

Article Snippet: After the block with 5% BSA, membranes were incubated overnight at 4 °C with primary antibodies against the following targets: Ube2s (Novus Biologicals, 1:500), HIF-1α (Invitrogen, 1:2000), Bax (Abcam, 1:1000), Bcl-2 (Abcam, 1:1000), β-Catenin (Cell Signaling, 1:500) and β-Actin (Santa Cruz, 1:5000).

Techniques: Activation Assay, Infection, Expressing, Plasmid Preparation, Control, Transfection, Western Blot, Activity Assay

Journal: Aging (Albany NY)

Article Title: Ube2s-stabilized β-catenin protects against myocardial ischemia/reperfusion injury by activating HIF-1α signaling

doi: 10.18632/aging.102960

Figure Lengend Snippet: Ube2s stabilizes β-Catenin after MI/R injury. ( A ) C57BL/6 mice were intra-myocardially infected with lentivirus expressing vector control or Ube2s 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of β-Catenin in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images (left) and relative band intensity analysis (right) are presented. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01. ( B ) C57BL/6 mice were intra-myocardially transfected with control siRNA (siCtrl) or Ube2s siRNA (siUbe2s) 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of β-Catenin in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images (left) and relative band intensity analysis (right) are presented. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01. ( C ) The cardiomyocytes overexpressing empty vector or Ube2s were treated with cycloheximide (CHX) for increasing time periods as indicated. The protein expression of β-Catenin and Ube2s was determined by Western blotting analysis. β-Actin was used as a loading control. The representative band images (left) and relative band intensity analysis of β-Catenin (right) are presented. The half-life is depicted by dot line. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01. ( D ) The lysates of cardiomyocytes stably overexpressing empty vector or Ube2s were immunoprecipitated (IP) with β-Catenin antibody. The IP products were further analyzed by Western blotting to detect ubiquitin expression. The expression of β-Catenin and Ube2s in the input fraction is presented below. ( E ) The lysates of heart tissues from mice, intra-myocardially infected with lentivirus expressing vector control or Ube2s 48 h prior to MI/R surgery, were immunoprecipitated (IP) with β-Catenin antibody. The IP products were further analyzed by Western blotting to detect ubiquitin expression. The expression of β-Catenin and Ube2s in the input fraction is presented below.

Article Snippet: After the block with 5% BSA, membranes were incubated overnight at 4 °C with primary antibodies against the following targets: Ube2s (Novus Biologicals, 1:500), HIF-1α (Invitrogen, 1:2000), Bax (Abcam, 1:1000), Bcl-2 (Abcam, 1:1000), β-Catenin (Cell Signaling, 1:500) and β-Actin (Santa Cruz, 1:5000).

Techniques: Infection, Expressing, Plasmid Preparation, Control, Western Blot, Transfection, Stable Transfection, Immunoprecipitation, Ubiquitin Proteomics

Journal: Aging (Albany NY)

Article Title: Ube2s-stabilized β-catenin protects against myocardial ischemia/reperfusion injury by activating HIF-1α signaling

doi: 10.18632/aging.102960

Figure Lengend Snippet: β-Catenin knockdown abrogates Ube2s-augmented HIF-1α activation and diminishes Ube2s protective effect on MI/R injury. ( A – B ) C57BL/6 mice were intra-myocardially infected with lentivirus expressing vector control or Ube2s in combination with the transfection with control siRNA (siCtrl) or β-Catenin siRNA (siβ-Catenin) 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein expression of targets as indicated in the heart was analyzed by Western blotting. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images ( A ) and relative band intensity analysis ( B ) are presented. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01; NS, not significant. ( C ) Heart samples were harvested as described in ( A ). The supernatants of the homogenized heart samples were collected, and the caspase-3 activity was determined. The results are expressed as nmol AFC/h/mg protein. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01. ( D ) Heart samples were harvested as described in ( A ), and the mid-myocardial cross sections were prepared. The infarct size in the heart sections was quantified, and the results of percentage of size are shown. AAR/LVA, ratio of area at risk (AAR) to left ventricular area (LVA); IA/AAR, ratio of infarct area (IA) to AAR. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01; *, P < 0.01; NS, not significant. ( E ) C57BL/6 mice were treated as in ( A ). The samples of serum were collected and the level of creatine phosphokinase (CPK) was quantified. Data are mean ± SD. Data were compared using Student’s t -test. **, P < 0.01.

Article Snippet: After the block with 5% BSA, membranes were incubated overnight at 4 °C with primary antibodies against the following targets: Ube2s (Novus Biologicals, 1:500), HIF-1α (Invitrogen, 1:2000), Bax (Abcam, 1:1000), Bcl-2 (Abcam, 1:1000), β-Catenin (Cell Signaling, 1:500) and β-Actin (Santa Cruz, 1:5000).

Techniques: Knockdown, Activation Assay, Infection, Expressing, Plasmid Preparation, Control, Transfection, Western Blot, Activity Assay

Journal: Aging (Albany NY)

Article Title: Ube2s-stabilized β-catenin protects against myocardial ischemia/reperfusion injury by activating HIF-1α signaling

doi: 10.18632/aging.102960

Figure Lengend Snippet: Ube2s/β-catenin/HIF-1α axis protects against MI/R injury. Graphic description of this study. Ube2s promotes HIF-1α activation through stabilizing β-catenin via a mechanism of ubiquitination modification, whereby acting to protect against MI/R injury, including reducing myocardial apoptosis, infarction and CPK release.

Article Snippet: After the block with 5% BSA, membranes were incubated overnight at 4 °C with primary antibodies against the following targets: Ube2s (Novus Biologicals, 1:500), HIF-1α (Invitrogen, 1:2000), Bax (Abcam, 1:1000), Bcl-2 (Abcam, 1:1000), β-Catenin (Cell Signaling, 1:500) and β-Actin (Santa Cruz, 1:5000).

Techniques: Activation Assay, Ubiquitin Proteomics, Modification

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 1. Immunostaining of Ube2S in normal lung tissues and NSCLC tissues. Bronchial epithelial cells (black arrow) and alveolar cells (grey arrow) show negative immunostaining for Ube2S (A). Bronchial epithelial cells (black arrow) and submucosal glands (grey arrow) show negative immunostaining for Ube2S (B). Weak and focal immunostaining of Ube2S is evident in some bronchial epithelial cells (C) and submucosal glands (D). Strong immunostaining of Ube2S is present in the cytoplasm and nuclei of squamous cell carcinoma (E) and adenocarcinoma (F) cells. (A: ×100; B, D, F: ×200; C, E: ×400)

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: Immunostaining

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 2. Survival function. Ube2S expression in non-small cell lung cancer is significantly associated with poor patient survival (34.9 ± 5.5 versus 56.4 ± 7.3 months) (Log rank test, p < 0.05)

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: Expressing

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 3. The function of Ube2S in lung cancer cells. Western blots show that Ube2S is expressed in bronchial epithelial HBE cells, and lung cancer A549 and NCI-H1299 cells (A). The MTT assay shows that overexpression of Ube2S in A549 cells significantly promotes cancer cell proliferation (B) (p < 0.05). The wound scratch healing assay shows that overexpression of Ube2S in A549 cells significantly promotes cancer cell migration (C) (*p < 0.05)

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: Western Blot, MTT Assay, Over Expression, Migration

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 4. Ube2S regulates Wnt/β-catenin signaling molecules and activity. Western blots show that overexpression of Ube2S in A549 cells significantly upregulates Wnt/β-catenin signaling molecules including β-catenin, cyclin D1, and MMP7 (A) (p < 0.05). The luciferase assay shows that overexpression of Ube2S in A549 cells significantly upregulates Wnt/β-catenin signaling activity (B) (p < 0.05)

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: Activity Assay, Western Blot, Over Expression, Luciferase

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 5. Wnt/β-catenin signaling inhibitor abolished the function of Ube2s in lung cancer cells. The MTT assay shows that addition of the Wnt/β-catenin signaling inhibitor, ETC-159, significantly inhibits the ability of Ube2S to promote cancer cell proliferation

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: MTT Assay

Journal: Molecular Biology of the Cell

Article Title: Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

doi: 10.1091/mbc.E15-02-0102

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: UBE2S , Abnova (PAB1701) , , 1:1000.

Techniques: Purification