Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: The Frequency of the SARS-CoV-2 S Protein D614G Variant over the Course of the Pandemic Has Increased Nearly to Fixation (A) The global frequency of the S protein D614G variant over time in the GISAID SARS-CoV-2 database as of June 25, 2020. The filled blue plot represents a seven-day rolling average of the fraction of sequences bearing the D614G variant for each collection date. Dates without published sequences are linearly interpolated. The overlaid black line shows the cumulative frequency of D614G in sequences collected up to and including each date. (B) The frequency of the D614G variant over time (blue) in sequences collected from six continental regions, using the same dataset as in (A), plotted as a seven-day rolling average. The frequency of the last date with data is carried forward where recent dates lack data to indicate the most recent calculated frequency (light blue). Red bars show the number of sequences that were used to provide the denominator for calculating the frequency for each date.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Variant Assay

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: SARS-CoV-2 D614G S Protein Variant Enhances Infectivity of Pseudotyped Lentiviruses in Cell Culture (A) Lentiviral virions bearing either GFP or Luciferase transgenes, and pseudotyped with either SARS-CoV-2 D614 or D614G S proteins, were produced by transfection of HEK293 cells and used to transduce human Calu3 lung cells, Caco2 colon cells, and either HEK293 or SupT1 cells stably expressing ACE2 and TMPRSS2. Relative infectivity of D614G versus D614, with D614 set at one, was determined based on flow cytometry for percent GFP positivity or on bulk luciferase activity. Each point represents the mean ± SD for transduction with lentiviral stocks derived from independent transfections, each value of which is the mean of three technical replicates. P values are the ratio paired t test (two-tailed). (B) Lentiviral virions bearing a luciferase transgene, pseudotyped with either SARS-CoV-2 D614 or D614G S proteins, were produced by transfection of HEK293 cells and used to transduce human HEK293 cells transiently transfected with plasmids encoding the indicated ACE2 orthologs. Relative infectivity of D614G versus D614, with D614 set at one, was determined based on bulk luciferase activity. Each point represents the mean ± SD after transduction by using lentiviral stock derived from an independent transfection, each of which is the mean of three technical replicates. P values are ratio paired t test (two-tailed).

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Variant Assay, Infection, Cell Culture, Luciferase, Produced, Transfection, Transduction, Stable Transfection, Expressing, Flow Cytometry, Activity Assay, Derivative Assay, Two Tailed Test

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: SARS-CoV-2 D614G S Protein Variant Binds ACE2 Weaker than the Ancestral Protein (A–D) SPR measurement of D614-ACE2 binding ([A] and [C]) and D614G-ACE2 binding ([B] and [D]) at 25°C ([A] and [B]) or 37°C ([C] and [D]). (E) Summary of kinetic parameters measured in (A)–(D). D614G binds ACE2 5-fold weaker than D614 at both temperatures tested.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Variant Assay, Binding Assay

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Neutralization Potency of Monoclonal Antibodies Targeting the SARS-CoV-2 S Protein Receptor-Binding Domain Is Not Attenuated by D614G (A and B) Vero cells were challenged with pVSV-SARS-CoV-2-S-mNeon pseudoparticles encoding either D614 (A) or D614G (B) S protein variants in the presence of serial dilutions of the indicated human monoclonal antibodies targeting the SARS-CoV-2 S protein receptor-binding domain or IgG1 isotype control. mNeon protein fluorescence was measured 24 h post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus only infection control. Data represent the mean ± SD of three technical replicates. (C) Neutralization potency (IC 50 ) of individual monoclonals and of combinations of monoclonals, against the SARS-CoV-2 D614G and D614G S protein variants, as indicated.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Neutralization, Bioprocessing, Binding Assay, Control, Fluorescence, Infection, Virus

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Summary of Cryo-EM Data Collection, Three-Dimensional Reconstruction, and Model Refinement

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Imaging

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Structural Determination of Spike D614G, Related to Figure 6 (A) Size exclusion column elution profiles of D614G (blue line) and D614 (red line). (B) Coomassie-stained SDS-PAGE of the peaks collected in (A) shows equivalent, full-length monomeric S proteins at around 180 kD. (C) A still frame from a raw micrograph movie. Individual particles can be clearly visualized. (D) 2D-clustering of trimeric D614G particles. Dagger denotes a top-view. Asterisk denotes a side-view. (E) Fourier shell correlation diagram for D614G (unmasked). (F) Half map Fourier shell correlation diagram (unmasked). (G) Model to map Fourier shell correlation diagram. (H) Local resolution of the ensemble map for Spike D614G.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Staining, SDS Page

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Structural Determination of Spike D614G (A) D614G envelope from the three-dimensional reconstruction. EMDB: EMD-22301. (B) Published D614 envelope (EMD-21452). Arrows point to the density corresponding to the receptor-binding domains, which is missing in the corresponding positions in (A). (C) Domain arrangement of the SARS-CoV-2 S protein. (D) Atomic model for D614G without the receptor-binding domain. PDB: 6XS6 . (E and F) Comparison of the D614G S1 subunit with the closed conformation (E) and open conformation (F) of the D614 S1 subunit. Arrows indicate the relative movement of the S1 subunit of D614G. (G) Position of amino acid 614 on the S protein. (H and I) Substitution of Asp614 with glycine changes hydrogen bonding around residue 614. In the case of D614 (H), an inter-protomer hydrogen bond is detected. For D614G (I), the Asp614-Thr859 hydrogen bond is eliminated, and interaction with intradomain Ala647 is strengthened. See also and .

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Binding Assay, Comparison, Residue

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Structural Determination of Spike D614G, Related to Figure 6 (A) Representative density map detail of an alpha helix region. (B) Representative density map detail of a loop region. (C) Representative density map detail of a beta strand region. Note: the relative positioning between (B) and (C) is the same as in the real structural model. No cryo-EM density is observed in between the loop where Gly614 resides and the beta strand where Thr859 resides.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: D614G Populates More Open Conformations Than Does the Ancestral S Protein (A) Cryo-EM density maps of the two conformations of D614G protomer. The first is a closed conformation with a buried RBD. The second is an open conformation with the RBD standing up. (B) Atomic models for the closed (left) and open (right) conformations for the two D614G protomers shown in (A). (C) Comparison of the two D614G protomer S1 subunit conformations with the corresponding conformations of the D614 protomer S1 subunit. (D) The D614G S protein trimer adopts four conformations. In addition to the all-closed and one open conformation detected with the D614 S protein trimer, the D614G S protein trimer adopts two-open and three-open conformations.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet:

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Binding Assay, Virus, Recombinant, Transfection, Luciferase, Bradford Protein Assay, Plasmid Preparation, Software, Single Particle, Microscopy

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