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mouse monoclonal antibody against ace2 (Proteintech)

Name: mouse monoclonal antibody against ace2
Brand: Proteintech
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Proteintech mouse monoclonal antibody against ace2

Glycan profiles of the SARS-CoV-2 S1 and the <t>ACE2</t> receptor. (A) Schematic diagram illustrating the process of preparing antibody-overlay lectin microarrays. (B, C) Scanned images were obtained for the analysis of glycopatterns from the SARS-CoV-2-S1 (B) and ACE2 (C). HEK293-expressing recombinant proteins of S1 and ACE2 were incubated with lectin microarrays. Subsequently, the microarrays were incubated with biotin-labeled primary antibodies and Cy3-labeled streptavidin. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II and STL), bisected and bi-antennary N-glycans (PHA-E), oligo-mannose type N-glycans (ConA and HHL), fucosylation (PSA and LCA) and α-2,3 linked sialic acid (MAL-II) were marked with white frames. (D, E) Analysis of glycopatterns on S1 (D) and ACE2 (E). The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of main types of glycans was calculated by diverging the sum of the NFIs of the lectins that recognized this type of glycan by the total NFIs of all lectins. Blue square: GlcNAc; yellow circle: galactose; yellow square: GalNAc; green circle: mannose; red triangle: fucose; purple diamond: sialic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mouse Monoclonal Antibody Against Ace2, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk"

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

Journal: Journal of Advanced Research

doi: 10.1016/j.jare.2024.12.010

Glycan profiles of the SARS-CoV-2 S1 and the ACE2 receptor. (A) Schematic diagram illustrating the process of preparing antibody-overlay lectin microarrays. (B, C) Scanned images were obtained for the analysis of glycopatterns from the SARS-CoV-2-S1 (B) and ACE2 (C). HEK293-expressing recombinant proteins of S1 and ACE2 were incubated with lectin microarrays. Subsequently, the microarrays were incubated with biotin-labeled primary antibodies and Cy3-labeled streptavidin. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II and STL), bisected and bi-antennary N-glycans (PHA-E), oligo-mannose type N-glycans (ConA and HHL), fucosylation (PSA and LCA) and α-2,3 linked sialic acid (MAL-II) were marked with white frames. (D, E) Analysis of glycopatterns on S1 (D) and ACE2 (E). The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of main types of glycans was calculated by diverging the sum of the NFIs of the lectins that recognized this type of glycan by the total NFIs of all lectins. Blue square: GlcNAc; yellow circle: galactose; yellow square: GalNAc; green circle: mannose; red triangle: fucose; purple diamond: sialic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure Legend Snippet: Glycan profiles of the SARS-CoV-2 S1 and the ACE2 receptor. (A) Schematic diagram illustrating the process of preparing antibody-overlay lectin microarrays. (B, C) Scanned images were obtained for the analysis of glycopatterns from the SARS-CoV-2-S1 (B) and ACE2 (C). HEK293-expressing recombinant proteins of S1 and ACE2 were incubated with lectin microarrays. Subsequently, the microarrays were incubated with biotin-labeled primary antibodies and Cy3-labeled streptavidin. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II and STL), bisected and bi-antennary N-glycans (PHA-E), oligo-mannose type N-glycans (ConA and HHL), fucosylation (PSA and LCA) and α-2,3 linked sialic acid (MAL-II) were marked with white frames. (D, E) Analysis of glycopatterns on S1 (D) and ACE2 (E). The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of main types of glycans was calculated by diverging the sum of the NFIs of the lectins that recognized this type of glycan by the total NFIs of all lectins. Blue square: GlcNAc; yellow circle: galactose; yellow square: GalNAc; green circle: mannose; red triangle: fucose; purple diamond: sialic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Glycoproteomics, Expressing, Recombinant, Incubation, Labeling, Binding Assay

Role of N-glycans in the interaction between S1 and ACE2. (A) Schematic diagram illustrating the process of manufacturing the SRAS-CoV-2-related recombinant protein microarrays. (B, C) The N-glycans on S1 of SARS-CoV-2/1 and ACE2 were removed by PNGase F glycosidase. The roles of N-glycans in the interaction between the SARS-CoV-2-S1 /ACE2 (B) and the SARS-CoV-1-S1/ACE2 (C) were evaluated using protein microarrays. Statistical analysis of the relative fluorescence intensities was conducted by comparing the PNGase F-treated S1 and ACE2 to the intact glycosylated protein using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (D) MD simulation of the interaction between the trimeric S protein and ACE2. The distances between the N-glycosites and the center of the binding interface (represented by the green globule) within 50 Å were marked with red spheres. Other N-glycosites were marked with yellow spheres. (E) The interactions of glycans at specific sites and GRDs (marked with a red frame) may be involved in the binding of the S protein to ACE2. (F) MD simulated the interactions of glycans at specific sites and GRDs. The distances between the terminal glycans on these sites and the three GRDs on the ACE and S1 subunit were monitored during a 100 ns MD simulation. The distances of N546-GRD1, N322-GRD2, and N53-GRD2 fluctuated between 1 and 15 Å, while the distances of N343-GRD3 and N165-GRD3 fluctuated between 20 and 35 Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure Legend Snippet: Role of N-glycans in the interaction between S1 and ACE2. (A) Schematic diagram illustrating the process of manufacturing the SRAS-CoV-2-related recombinant protein microarrays. (B, C) The N-glycans on S1 of SARS-CoV-2/1 and ACE2 were removed by PNGase F glycosidase. The roles of N-glycans in the interaction between the SARS-CoV-2-S1 /ACE2 (B) and the SARS-CoV-1-S1/ACE2 (C) were evaluated using protein microarrays. Statistical analysis of the relative fluorescence intensities was conducted by comparing the PNGase F-treated S1 and ACE2 to the intact glycosylated protein using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (D) MD simulation of the interaction between the trimeric S protein and ACE2. The distances between the N-glycosites and the center of the binding interface (represented by the green globule) within 50 Å were marked with red spheres. Other N-glycosites were marked with yellow spheres. (E) The interactions of glycans at specific sites and GRDs (marked with a red frame) may be involved in the binding of the S protein to ACE2. (F) MD simulated the interactions of glycans at specific sites and GRDs. The distances between the terminal glycans on these sites and the three GRDs on the ACE and S1 subunit were monitored during a 100 ns MD simulation. The distances of N546-GRD1, N322-GRD2, and N53-GRD2 fluctuated between 1 and 15 Å, while the distances of N343-GRD3 and N165-GRD3 fluctuated between 20 and 35 Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Recombinant, Fluorescence, Binding Assay

β1-4 galactosylated N-glycans of ACE2 mediated the binding of S1 of SARS-CoV-2 and its variants. (A) Molecular docking analysis of S1 and ACE2 with various saccharides. The potential binding capacities of S1 of SARS-CoV-2 (Wuhan-Hu-1 strain, wild type) and its variants (Delta and Omicron), as well as ACE2, to various saccharides were predicted by molecular docking analysis. The saccharides were listed in columns, S1 and ACE2 were listed in rows. The different binding abilities were represented by the values of binding free energy, which were indicated by the color of each square: red: high affinity, blue: low affinity, Xyl: xylose, Glc: glucose; Man: mannose; GlcNAc: N-acetylglucosamine, GalNAc: N-acetylgalactosamine; SA: sialic acid. (B) Validation of β1-4 galactosylation level in intact and de-β1-4galactosylated ACE2. After β1-4 galactosidase treatment, the level of β1-4 galactosylation on ACE2 was detected by lectin blotting of MAL-I. The protein level of ACE2 served as the control. (C) Scanning images of protein microarrays incubated with 1 μg of intact or de-β1-4galactosylated ACE2. (D) Effect of β1-4 galactosylation of ACE2 on the binding of S1 to ACE2. The relative fluorescence intensities were statistically analyzed by comparing the de-β1-4galactosylated ACE2 to intact ACE2 using an unpaired t test with Welch's correction. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure Legend Snippet: β1-4 galactosylated N-glycans of ACE2 mediated the binding of S1 of SARS-CoV-2 and its variants. (A) Molecular docking analysis of S1 and ACE2 with various saccharides. The potential binding capacities of S1 of SARS-CoV-2 (Wuhan-Hu-1 strain, wild type) and its variants (Delta and Omicron), as well as ACE2, to various saccharides were predicted by molecular docking analysis. The saccharides were listed in columns, S1 and ACE2 were listed in rows. The different binding abilities were represented by the values of binding free energy, which were indicated by the color of each square: red: high affinity, blue: low affinity, Xyl: xylose, Glc: glucose; Man: mannose; GlcNAc: N-acetylglucosamine, GalNAc: N-acetylgalactosamine; SA: sialic acid. (B) Validation of β1-4 galactosylation level in intact and de-β1-4galactosylated ACE2. After β1-4 galactosidase treatment, the level of β1-4 galactosylation on ACE2 was detected by lectin blotting of MAL-I. The protein level of ACE2 served as the control. (C) Scanning images of protein microarrays incubated with 1 μg of intact or de-β1-4galactosylated ACE2. (D) Effect of β1-4 galactosylation of ACE2 on the binding of S1 to ACE2. The relative fluorescence intensities were statistically analyzed by comparing the de-β1-4galactosylated ACE2 to intact ACE2 using an unpaired t test with Welch's correction. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Binding Assay, Biomarker Discovery, Control, Incubation, Fluorescence

Evaluation of the ability of free saccharides to block S1 and ACE2 binding. (A, B) Scanning images of protein microarrays. ACE2 was mixed with GalNAc (A) or Galβ1-3GalNAc (B), and the inhibitory effect of saccharides was evaluated using protein microarrays. (C, D) Effect of GalNAc (C) and Galβ-1,3GalNAc (D) on the interaction between S1 of SARS-CoV-2/1 and ACE2. The binding signals were extracted, and the relative fluorescence intensities were compared with those of the controls using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated.

Figure Legend Snippet: Evaluation of the ability of free saccharides to block S1 and ACE2 binding. (A, B) Scanning images of protein microarrays. ACE2 was mixed with GalNAc (A) or Galβ1-3GalNAc (B), and the inhibitory effect of saccharides was evaluated using protein microarrays. (C, D) Effect of GalNAc (C) and Galβ-1,3GalNAc (D) on the interaction between S1 of SARS-CoV-2/1 and ACE2. The binding signals were extracted, and the relative fluorescence intensities were compared with those of the controls using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated.

Techniques Used: Blocking Assay, Binding Assay, Fluorescence

Evaluation of isolated glycoproteins for the inhibition of S1 and ACE2 binding. (A) The scanned image was obtained from the lectin microarray analysis of glycoproteins isolated from bovine milk. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II), bisected N-glycans (PHA-E), high-mannose glycans (ConA), fucosylation (AAL, PSA, and LCA), α2-3 linked sialic acid (MAL-II), and α2-6 linked sialic acid (SNA) were marked with white frames. (B) Analysis of glycopatterns on isolated glycoproteins. The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of galactosylated glycans was calculated by diverging the sum of the NFIs of the lectins that recognized Gal/GalNAc by the total NFIs. (C, D) Evaluation of the effect of intact and de-sialylated isolated glycoproteins on the interaction between S1 of SARS-CoV-2/1 and ACE2. The intact isolated glycoproteins (C) or de-sialylated isolated glycoproteins (D) were mixed with ACE2 and incubated with protein microarrays. The relative binding intensities of each group were compared with those of the control group, and any significant differences between groups were determined using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (E) Inhibition curves for intact isolated glycoproteins (upper) and de-sialylated isolated glycoproteins (lower). Four-parameter inhibition curves were generated, and the particular IC50 values for intact isolated glycoproteins and de-sialylated isolated glycoproteins were indicated in this graph. The data were obtained from three biological replicates and presented as the mean ± SD (error bars).

Figure Legend Snippet: Evaluation of isolated glycoproteins for the inhibition of S1 and ACE2 binding. (A) The scanned image was obtained from the lectin microarray analysis of glycoproteins isolated from bovine milk. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II), bisected N-glycans (PHA-E), high-mannose glycans (ConA), fucosylation (AAL, PSA, and LCA), α2-3 linked sialic acid (MAL-II), and α2-6 linked sialic acid (SNA) were marked with white frames. (B) Analysis of glycopatterns on isolated glycoproteins. The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of galactosylated glycans was calculated by diverging the sum of the NFIs of the lectins that recognized Gal/GalNAc by the total NFIs. (C, D) Evaluation of the effect of intact and de-sialylated isolated glycoproteins on the interaction between S1 of SARS-CoV-2/1 and ACE2. The intact isolated glycoproteins (C) or de-sialylated isolated glycoproteins (D) were mixed with ACE2 and incubated with protein microarrays. The relative binding intensities of each group were compared with those of the control group, and any significant differences between groups were determined using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (E) Inhibition curves for intact isolated glycoproteins (upper) and de-sialylated isolated glycoproteins (lower). Four-parameter inhibition curves were generated, and the particular IC50 values for intact isolated glycoproteins and de-sialylated isolated glycoproteins were indicated in this graph. The data were obtained from three biological replicates and presented as the mean ± SD (error bars).

Techniques Used: Isolation, Inhibition, Binding Assay, Microarray, Glycoproteomics, Incubation, Control, Generated

Image Search Results

FIGURE 1 In‐house and commercial ACE2 enzymatic immunoassay (EIA) results of pre‐COVID‐19 donor control sera, COVID‐19 convalescent patient, and vaccine recipient sera. (A, B) IgM EIA results of COVID‐19 convalescent sera classified based on severity. (C, D) IgG EIA results of COVID‐19 convalescent sera classified based on severity. (E, F) IgG EIA results of COVID‐19 vaccine recipients based on type of vaccine. Bars represent median and interquartile range. Intergroup comparisons of medians were performed using Dunn's multiple comparisons test. Ns: not significant; *p ≤0.05; ***p ≤0.001; ****p ≤0.0001. ACE2, angiotensin‐converting enzyme 2; COVID‐19, coronavirus disease 2019.

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 1 In‐house and commercial ACE2 enzymatic immunoassay (EIA) results of pre‐COVID‐19 donor control sera, COVID‐19 convalescent patient, and vaccine recipient sera. (A, B) IgM EIA results of COVID‐19 convalescent sera classified based on severity. (C, D) IgG EIA results of COVID‐19 convalescent sera classified based on severity. (E, F) IgG EIA results of COVID‐19 vaccine recipients based on type of vaccine. Bars represent median and interquartile range. Intergroup comparisons of medians were performed using Dunn's multiple comparisons test. Ns: not significant; *p ≤0.05; ***p ≤0.001; ****p ≤0.0001. ACE2, angiotensin‐converting enzyme 2; COVID‐19, coronavirus disease 2019.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques: Enzyme Immunoassay, Control

FIGURE 2 Correlations between ACE2 IgG enzymatic immunoassay optical densities (OD) and surrogate neutralizing antibody levels of CoronaVac (A, B) and Comirnaty (C, D) cohorts using commercial and in‐house ACE2 peptides. Strength of correlation was assessed using Spearman's rank correlation. ACE2, angiotensin‐converting enzyme 2.

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 2 Correlations between ACE2 IgG enzymatic immunoassay optical densities (OD) and surrogate neutralizing antibody levels of CoronaVac (A, B) and Comirnaty (C, D) cohorts using commercial and in‐house ACE2 peptides. Strength of correlation was assessed using Spearman's rank correlation. ACE2, angiotensin‐converting enzyme 2.

Techniques: Enzyme Immunoassay

FIGURE 3 Trends of ACE2 IgG optical densities (ODs) using in‐house (A) and commercial (B) peptides for vaccine recipients testing positive at Day 56 post‐first dose. Each line represents trend for individual recipients. SNV020, SNV027, and SNV058 are CoronaVac recipients. BNT007, BNT012, BNT032, BNT081, BNT090, and BNT092 are Comirnaty recipients. The second timepoint is either Day 21 (for Comirnaty recipients) or Day 28 (for CoronaVac recipients). ACE2, angiotensin‐converting enzyme 2.

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 3 Trends of ACE2 IgG optical densities (ODs) using in‐house (A) and commercial (B) peptides for vaccine recipients testing positive at Day 56 post‐first dose. Each line represents trend for individual recipients. SNV020, SNV027, and SNV058 are CoronaVac recipients. BNT007, BNT012, BNT032, BNT081, BNT090, and BNT092 are Comirnaty recipients. The second timepoint is either Day 21 (for Comirnaty recipients) or Day 28 (for CoronaVac recipients). ACE2, angiotensin‐converting enzyme 2.

Techniques:

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Glycan profiles of the SARS-CoV-2 S1 and the ACE2 receptor. (A) Schematic diagram illustrating the process of preparing antibody-overlay lectin microarrays. (B, C) Scanned images were obtained for the analysis of glycopatterns from the SARS-CoV-2-S1 (B) and ACE2 (C). HEK293-expressing recombinant proteins of S1 and ACE2 were incubated with lectin microarrays. Subsequently, the microarrays were incubated with biotin-labeled primary antibodies and Cy3-labeled streptavidin. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II and STL), bisected and bi-antennary N-glycans (PHA-E), oligo-mannose type N-glycans (ConA and HHL), fucosylation (PSA and LCA) and α-2,3 linked sialic acid (MAL-II) were marked with white frames. (D, E) Analysis of glycopatterns on S1 (D) and ACE2 (E). The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of main types of glycans was calculated by diverging the sum of the NFIs of the lectins that recognized this type of glycan by the total NFIs of all lectins. Blue square: GlcNAc; yellow circle: galactose; yellow square: GalNAc; green circle: mannose; red triangle: fucose; purple diamond: sialic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Glycoproteomics, Expressing, Recombinant, Incubation, Labeling, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Role of N-glycans in the interaction between S1 and ACE2. (A) Schematic diagram illustrating the process of manufacturing the SRAS-CoV-2-related recombinant protein microarrays. (B, C) The N-glycans on S1 of SARS-CoV-2/1 and ACE2 were removed by PNGase F glycosidase. The roles of N-glycans in the interaction between the SARS-CoV-2-S1 /ACE2 (B) and the SARS-CoV-1-S1/ACE2 (C) were evaluated using protein microarrays. Statistical analysis of the relative fluorescence intensities was conducted by comparing the PNGase F-treated S1 and ACE2 to the intact glycosylated protein using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (D) MD simulation of the interaction between the trimeric S protein and ACE2. The distances between the N-glycosites and the center of the binding interface (represented by the green globule) within 50 Å were marked with red spheres. Other N-glycosites were marked with yellow spheres. (E) The interactions of glycans at specific sites and GRDs (marked with a red frame) may be involved in the binding of the S protein to ACE2. (F) MD simulated the interactions of glycans at specific sites and GRDs. The distances between the terminal glycans on these sites and the three GRDs on the ACE and S1 subunit were monitored during a 100 ns MD simulation. The distances of N546-GRD1, N322-GRD2, and N53-GRD2 fluctuated between 1 and 15 Å, while the distances of N343-GRD3 and N165-GRD3 fluctuated between 20 and 35 Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Techniques: Recombinant, Fluorescence, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: β1-4 galactosylated N-glycans of ACE2 mediated the binding of S1 of SARS-CoV-2 and its variants. (A) Molecular docking analysis of S1 and ACE2 with various saccharides. The potential binding capacities of S1 of SARS-CoV-2 (Wuhan-Hu-1 strain, wild type) and its variants (Delta and Omicron), as well as ACE2, to various saccharides were predicted by molecular docking analysis. The saccharides were listed in columns, S1 and ACE2 were listed in rows. The different binding abilities were represented by the values of binding free energy, which were indicated by the color of each square: red: high affinity, blue: low affinity, Xyl: xylose, Glc: glucose; Man: mannose; GlcNAc: N-acetylglucosamine, GalNAc: N-acetylgalactosamine; SA: sialic acid. (B) Validation of β1-4 galactosylation level in intact and de-β1-4galactosylated ACE2. After β1-4 galactosidase treatment, the level of β1-4 galactosylation on ACE2 was detected by lectin blotting of MAL-I. The protein level of ACE2 served as the control. (C) Scanning images of protein microarrays incubated with 1 μg of intact or de-β1-4galactosylated ACE2. (D) Effect of β1-4 galactosylation of ACE2 on the binding of S1 to ACE2. The relative fluorescence intensities were statistically analyzed by comparing the de-β1-4galactosylated ACE2 to intact ACE2 using an unpaired t test with Welch's correction. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Techniques: Binding Assay, Biomarker Discovery, Control, Incubation, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of the ability of free saccharides to block S1 and ACE2 binding. (A, B) Scanning images of protein microarrays. ACE2 was mixed with GalNAc (A) or Galβ1-3GalNAc (B), and the inhibitory effect of saccharides was evaluated using protein microarrays. (C, D) Effect of GalNAc (C) and Galβ-1,3GalNAc (D) on the interaction between S1 of SARS-CoV-2/1 and ACE2. The binding signals were extracted, and the relative fluorescence intensities were compared with those of the controls using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated.

Techniques: Blocking Assay, Binding Assay, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of isolated glycoproteins for the inhibition of S1 and ACE2 binding. (A) The scanned image was obtained from the lectin microarray analysis of glycoproteins isolated from bovine milk. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II), bisected N-glycans (PHA-E), high-mannose glycans (ConA), fucosylation (AAL, PSA, and LCA), α2-3 linked sialic acid (MAL-II), and α2-6 linked sialic acid (SNA) were marked with white frames. (B) Analysis of glycopatterns on isolated glycoproteins. The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of galactosylated glycans was calculated by diverging the sum of the NFIs of the lectins that recognized Gal/GalNAc by the total NFIs. (C, D) Evaluation of the effect of intact and de-sialylated isolated glycoproteins on the interaction between S1 of SARS-CoV-2/1 and ACE2. The intact isolated glycoproteins (C) or de-sialylated isolated glycoproteins (D) were mixed with ACE2 and incubated with protein microarrays. The relative binding intensities of each group were compared with those of the control group, and any significant differences between groups were determined using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (E) Inhibition curves for intact isolated glycoproteins (upper) and de-sialylated isolated glycoproteins (lower). Four-parameter inhibition curves were generated, and the particular IC50 values for intact isolated glycoproteins and de-sialylated isolated glycoproteins were indicated in this graph. The data were obtained from three biological replicates and presented as the mean ± SD (error bars).

Techniques: Isolation, Inhibition, Binding Assay, Microarray, Glycoproteomics, Incubation, Control, Generated

Features of primer sequences for real-team PCR expression analysis.

Journal: Antioxidants

Article Title: An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

doi: 10.3390/antiox11040764

Figure Lengend Snippet: Features of primer sequences for real-team PCR expression analysis.

Article Snippet: Blots were blocked in TBS-T containing 5% non-fat dry milk and incubated overnight at 4 °C with either mouse monoclonal antibody against ACE2 (cat# Sc-73668; dilution 1:500), or rabbit polyclonal antibodies directed against Akt1/2/3 (Santa Cruz Biotechnology, cat# Sc-8312), pAkt1/2/3-Ser473 (cat# Sc-7985-R), AMPKα (Cell Signaling Technology, cat# 5831; dil 1:500), pAMPKα (Thr172) (Cell Signaling Technology, cat# 2535; dil 1:500), eNOS (cat# N3893), or goat polyclonal antibody directed against pNOS3-Ser1177 (cat# Sc-12972; dil. 1:500).

Techniques: Expressing

( A ) ace2 mRNA expression levels in goldfish C. auratus tissues. The amounts of target mRNA are calculated as 2-ΔCt mean values obtained from the output Ct values of two rounds of real-time PCR assays for each of three independent biological replicates. Statistics were assessed by one-way ANOVA followed by a Sidak’s multiple comparison test (**** p < 0.0001); ( B ) Representative immunoblotting of ACE2 expression in the goldfish heart. M: marker; H: heart; ( C ) Representative HPLC chromatogram showing Alamandine (Ala) elution from C. auratus plasma compared to Alamandine standard (red dotted line).

Journal: Antioxidants

Article Title: An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

doi: 10.3390/antiox11040764

Figure Lengend Snippet: ( A ) ace2 mRNA expression levels in goldfish C. auratus tissues. The amounts of target mRNA are calculated as 2-ΔCt mean values obtained from the output Ct values of two rounds of real-time PCR assays for each of three independent biological replicates. Statistics were assessed by one-way ANOVA followed by a Sidak’s multiple comparison test (**** p < 0.0001); ( B ) Representative immunoblotting of ACE2 expression in the goldfish heart. M: marker; H: heart; ( C ) Representative HPLC chromatogram showing Alamandine (Ala) elution from C. auratus plasma compared to Alamandine standard (red dotted line).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison, Western Blot, Marker, Clinical Proteomics

( A ) Quantitative real-time PCR analysis of ace2 mRNA expression in cardiac extracts of goldfish C. auratus exposed to normoxia and hypoxia. Comparison of 2-ΔCt mean values of normoxic and hypoxic hearts, reported as percent fold change ( y -axis). Statistic was assessed by one-way ANOVA followed by a Sidak’s multiple comparison test ( n = 3); ( B ) Representative immunoblot and densitometric analysis of ACE2 expression in cardiac extracts of goldfish C. auratus exposed to normoxia and hypoxia. Data were expressed as means ± s.e.m. of absolute values from individual experiments ( n = 3). Statistical analysis was performed by two-tailed unpaired t -test (* p < 0.05). ( C ) Representative HPLC chromatogram showing Alamandine (Ala) elution from plasma samples of goldfish C. auratus exposed to hypoxia compared with standard (red dotted line).

Journal: Antioxidants

Article Title: An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

doi: 10.3390/antiox11040764

Figure Lengend Snippet: ( A ) Quantitative real-time PCR analysis of ace2 mRNA expression in cardiac extracts of goldfish C. auratus exposed to normoxia and hypoxia. Comparison of 2-ΔCt mean values of normoxic and hypoxic hearts, reported as percent fold change ( y -axis). Statistic was assessed by one-way ANOVA followed by a Sidak’s multiple comparison test ( n = 3); ( B ) Representative immunoblot and densitometric analysis of ACE2 expression in cardiac extracts of goldfish C. auratus exposed to normoxia and hypoxia. Data were expressed as means ± s.e.m. of absolute values from individual experiments ( n = 3). Statistical analysis was performed by two-tailed unpaired t -test (* p < 0.05). ( C ) Representative HPLC chromatogram showing Alamandine (Ala) elution from plasma samples of goldfish C. auratus exposed to hypoxia compared with standard (red dotted line).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Comparison, Western Blot, Two Tailed Test, Clinical Proteomics

Analysis of the protein–protein interaction network (PPI) by the STRING online suite (version 11.5). The PPI network includes 14 proteins (number of nodes: 14; number of edges: 38; average node degree: 5.43; avg. local clustering coefficient: 0.623; expected number of edges: 2; PPI enrichment p -value: <1.0 × 10 −16 ). ace2-related proteins: ace2, Angiotensin I converting enzyme 2; agtr2, Angiotensin II receptor, type 2; ace, Angiotensin I converting enzyme 1; agtr1a, Angiotensin II receptor, type 1a; agtr1b, Angiotensin II receptor, type 1b; ren, Renin; nos1, nitric oxide synthase; agt, Angiotensinogen; hif1-related proteins: egln1a, Egl-9 family hypoxia-inducible factor 1; hif1an, Hypoxia-inducible factor 1-alpha inhibitor; tceb1b, Transcription elongation factor B (SIII), polypeptide 1b; hif1al, Hypoxia-inducible factor 1, alpha subunit, -like; hif1ab, Hypoxia-inducible factor 1, alpha subunit b; hif1aa, Hypoxia-inducible factor 1, alpha subunit a.

Journal: Antioxidants

Article Title: An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

doi: 10.3390/antiox11040764

Figure Lengend Snippet: Analysis of the protein–protein interaction network (PPI) by the STRING online suite (version 11.5). The PPI network includes 14 proteins (number of nodes: 14; number of edges: 38; average node degree: 5.43; avg. local clustering coefficient: 0.623; expected number of edges: 2; PPI enrichment p -value: <1.0 × 10 −16 ). ace2-related proteins: ace2, Angiotensin I converting enzyme 2; agtr2, Angiotensin II receptor, type 2; ace, Angiotensin I converting enzyme 1; agtr1a, Angiotensin II receptor, type 1a; agtr1b, Angiotensin II receptor, type 1b; ren, Renin; nos1, nitric oxide synthase; agt, Angiotensinogen; hif1-related proteins: egln1a, Egl-9 family hypoxia-inducible factor 1; hif1an, Hypoxia-inducible factor 1-alpha inhibitor; tceb1b, Transcription elongation factor B (SIII), polypeptide 1b; hif1al, Hypoxia-inducible factor 1, alpha subunit, -like; hif1ab, Hypoxia-inducible factor 1, alpha subunit b; hif1aa, Hypoxia-inducible factor 1, alpha subunit a.

Techniques:

Figure 1. (a) The life cycle of SARS-CoV-2 virus: (1) SARS-CoV-2 attaches to the surface of a human cell via interactions between S protein and ACE2 receptor. (2) After membrane fusion, it undergoes endocytosis and diﬀuses into the inner space of the human cell. (3) In the uncoating process, the RNA of SARS-CoV-2 is released into cytoplasm of human cell. (4) Translation of RNA takes places with the aid of a series of proteins. (5) RNA synthesis and structural protein translation lead to the assembling of a new virus. (6) The matured new virus leaves the host cell via exocytosis. Several compounds were used to treat SARS-CoV-2 via diﬀerent mechanisms. Both chloroquine and hydroxychloroquine were used to stop uncoating; lopinavir was used to stop proteolysis; and both remdesivir and favipiravir were used to stop RNA synthesis. (b) Structural changes

Journal: ACS pharmacology & translational science

Article Title: Clinical HDAC Inhibitors Are Effective Drugs to Prevent the Entry of SARS-CoV2.

doi: 10.1021/acsptsci.0c00163

Figure Lengend Snippet: Figure 1. (a) The life cycle of SARS-CoV-2 virus: (1) SARS-CoV-2 attaches to the surface of a human cell via interactions between S protein and ACE2 receptor. (2) After membrane fusion, it undergoes endocytosis and diﬀuses into the inner space of the human cell. (3) In the uncoating process, the RNA of SARS-CoV-2 is released into cytoplasm of human cell. (4) Translation of RNA takes places with the aid of a series of proteins. (5) RNA synthesis and structural protein translation lead to the assembling of a new virus. (6) The matured new virus leaves the host cell via exocytosis. Several compounds were used to treat SARS-CoV-2 via diﬀerent mechanisms. Both chloroquine and hydroxychloroquine were used to stop uncoating; lopinavir was used to stop proteolysis; and both remdesivir and favipiravir were used to stop RNA synthesis. (b) Structural changes

Article Snippet: Mouse monoclonal antibody against beta-actin (sc-47778) and mouse monoclonal antibody against human ACE2 (sc390851) were purchased from Santa Cruz Biotechnology (Dallas, USA).

Techniques: Virus, Membrane

Figure 2. HDAC clinical inhibitors prevent SARS-CoV-2 cell entry. (a) Screen for clinical drugs that can eﬃciently inhibit host cell entry of SARS- 2-S pseudotyped particles. ACE2-GFP stably expressing 293T cells were preincubated with indicated concentrations of drugs for 2 h and subsequently inoculated with SARS-2-S pseudotyped particles for an additional 3 h. The medium with pseudotyped particles was then removed, and cells were recultured in fresh medium for 40 h. Luciferase activity assay was conducted to analyze the virus entry eﬃciency. (b) Inhibition of SARS-2-S pseudotyped viruses entry into ACE2-GFP transfected 293T cells. 293T cells transfected with ACE2-GFP for 24 h were preincubated with indicated drugs for 1 h, and then further inoculated with SARS-2-S pseudotyped viruses for additional 2 h in the presence of drugs. The cell entry of SARS-2-S pseudotyped viruses were examined by immunoﬂuorescence staining using anti-FLAG antibodies. (c,d), Concentration- and time-dependent inhibitory eﬀects of romidepsin on host cell entry of SARS-2-S pseudotyped particles. 293T cells transfected with ACE2-GFP were treated with increased doses of romidepsin (c) or diﬀerent time of drug treatment (d). The cell entry of SARS-2-S pseudotyped viruses were detected by anti-FLAG staining. (e) Immunoﬂuorescence staining shows that host cell entry of SARS-S pseudotyped particles was blocked by romidepsin. 293T cells transfected with ACE2-GFP were treated as in (b) with the replacement of SARS-2-S pseudotyped particles as SARS-S pseudotyped particles. The anti-FLAG immunoﬂuorescence staining images were as shown. (f) Luciferase activity assay shows that host cell (ACE2-GFP stably expressing 293T) entry of SARS-S pseudotyped particles was blocked by romidepsin. ACE2-GFP stably expressing 293T cells were preincubated for 1 h with an increased dose of Romidepsin, then inoculated with SARS-S pseudotyped viruses for an additional 3 h, followed by Luciferase activity assay as in panel a at 40 h postinoculation. For luciferase activity assay in panels a and f, the average value of three independent experiments was calculated with triplicate samples. Error bars indicate SEM. Statistical signiﬁcance was tested by two-way ANOVA with Dunnett post-test. Groups of cells with no drug treatment were used as controls.

Journal: ACS pharmacology & translational science

Article Title: Clinical HDAC Inhibitors Are Effective Drugs to Prevent the Entry of SARS-CoV2.

doi: 10.1021/acsptsci.0c00163

Figure Lengend Snippet: Figure 2. HDAC clinical inhibitors prevent SARS-CoV-2 cell entry. (a) Screen for clinical drugs that can eﬃciently inhibit host cell entry of SARS- 2-S pseudotyped particles. ACE2-GFP stably expressing 293T cells were preincubated with indicated concentrations of drugs for 2 h and subsequently inoculated with SARS-2-S pseudotyped particles for an additional 3 h. The medium with pseudotyped particles was then removed, and cells were recultured in fresh medium for 40 h. Luciferase activity assay was conducted to analyze the virus entry eﬃciency. (b) Inhibition of SARS-2-S pseudotyped viruses entry into ACE2-GFP transfected 293T cells. 293T cells transfected with ACE2-GFP for 24 h were preincubated with indicated drugs for 1 h, and then further inoculated with SARS-2-S pseudotyped viruses for additional 2 h in the presence of drugs. The cell entry of SARS-2-S pseudotyped viruses were examined by immunoﬂuorescence staining using anti-FLAG antibodies. (c,d), Concentration- and time-dependent inhibitory eﬀects of romidepsin on host cell entry of SARS-2-S pseudotyped particles. 293T cells transfected with ACE2-GFP were treated with increased doses of romidepsin (c) or diﬀerent time of drug treatment (d). The cell entry of SARS-2-S pseudotyped viruses were detected by anti-FLAG staining. (e) Immunoﬂuorescence staining shows that host cell entry of SARS-S pseudotyped particles was blocked by romidepsin. 293T cells transfected with ACE2-GFP were treated as in (b) with the replacement of SARS-2-S pseudotyped particles as SARS-S pseudotyped particles. The anti-FLAG immunoﬂuorescence staining images were as shown. (f) Luciferase activity assay shows that host cell (ACE2-GFP stably expressing 293T) entry of SARS-S pseudotyped particles was blocked by romidepsin. ACE2-GFP stably expressing 293T cells were preincubated for 1 h with an increased dose of Romidepsin, then inoculated with SARS-S pseudotyped viruses for an additional 3 h, followed by Luciferase activity assay as in panel a at 40 h postinoculation. For luciferase activity assay in panels a and f, the average value of three independent experiments was calculated with triplicate samples. Error bars indicate SEM. Statistical signiﬁcance was tested by two-way ANOVA with Dunnett post-test. Groups of cells with no drug treatment were used as controls.

Article Snippet: Mouse monoclonal antibody against beta-actin (sc-47778) and mouse monoclonal antibody against human ACE2 (sc390851) were purchased from Santa Cruz Biotechnology (Dallas, USA).

Techniques: Stable Transfection, Expressing, Luciferase, Activity Assay, Virus, Inhibition, Transfection, Staining, Concentration Assay

Figure 4. Screen for clinically used HDAC inhibitors to suppress SARS-2-S-driven host cell entry. (a) Luciferase activity assay-based screening of clinically used HDAC inhibitors for suppressing SARS-2-S pseudovirus cell entry. ACE2-GFP stably expressed 293T cells were preincubated with the increased dose of indicated clinical drugs for 2 h and subsequently inoculated with SARS-2-S pseudotyped particles for 3 h, then further cultured for an additional 40 h with fresh medium. Luciferase activity in each cell lysate was examined to analyze the entry eﬃciency of SARS-2-S pseudovirus. The average value from three independent experiments with triplicate samples was calculated. Error bars indicate SEM. Statistical signiﬁcance was tested by two-way ANOVA with Dunnett post-test. Cells without drug treatment were used as controls. (b) Immunoﬂuorescence staining reveals that four out of 18 H drugs can eﬃciently inhibit cell entry of SARS-2-S pseudotype particles. ACE2-GFP transfected 293T cells were pretreated with the indicated drug for 1 h and subsequently inoculated with SARS-2-S pseudotyped particles for an additional 2 h. Cells were ﬁxed and stained with antibodies against FLAG to detect the intracellular SARS-2-S pseudovirus. Green, ACE2-GFP; red, FLAG; blue, DAPI. Scale bar is 5 μm. Quantiﬁcation of the ﬂuorescence intensity along the line across the perinuclear region was shown at the bottom. Green line, ACE2- GFP; red line, FLAG. The two-head arrows indicate the perinuclear region where the ACE2-GFP signal was highest. (c) Analysis of the distribution of internalized pseudotyped particles. Interspace that is within 2 μm from the cellular boundary is considered as the edge area. Percent ratio of edge signaling was calculated as edge area signal/total area signal × 100. Thirty cells from each group were used for quantiﬁcation.

Journal: ACS pharmacology & translational science

Article Title: Clinical HDAC Inhibitors Are Effective Drugs to Prevent the Entry of SARS-CoV2.

doi: 10.1021/acsptsci.0c00163

Figure Lengend Snippet: Figure 4. Screen for clinically used HDAC inhibitors to suppress SARS-2-S-driven host cell entry. (a) Luciferase activity assay-based screening of clinically used HDAC inhibitors for suppressing SARS-2-S pseudovirus cell entry. ACE2-GFP stably expressed 293T cells were preincubated with the increased dose of indicated clinical drugs for 2 h and subsequently inoculated with SARS-2-S pseudotyped particles for 3 h, then further cultured for an additional 40 h with fresh medium. Luciferase activity in each cell lysate was examined to analyze the entry eﬃciency of SARS-2-S pseudovirus. The average value from three independent experiments with triplicate samples was calculated. Error bars indicate SEM. Statistical signiﬁcance was tested by two-way ANOVA with Dunnett post-test. Cells without drug treatment were used as controls. (b) Immunoﬂuorescence staining reveals that four out of 18 H drugs can eﬃciently inhibit cell entry of SARS-2-S pseudotype particles. ACE2-GFP transfected 293T cells were pretreated with the indicated drug for 1 h and subsequently inoculated with SARS-2-S pseudotyped particles for an additional 2 h. Cells were ﬁxed and stained with antibodies against FLAG to detect the intracellular SARS-2-S pseudovirus. Green, ACE2-GFP; red, FLAG; blue, DAPI. Scale bar is 5 μm. Quantiﬁcation of the ﬂuorescence intensity along the line across the perinuclear region was shown at the bottom. Green line, ACE2- GFP; red line, FLAG. The two-head arrows indicate the perinuclear region where the ACE2-GFP signal was highest. (c) Analysis of the distribution of internalized pseudotyped particles. Interspace that is within 2 μm from the cellular boundary is considered as the edge area. Percent ratio of edge signaling was calculated as edge area signal/total area signal × 100. Thirty cells from each group were used for quantiﬁcation.

Article Snippet: Mouse monoclonal antibody against beta-actin (sc-47778) and mouse monoclonal antibody against human ACE2 (sc390851) were purchased from Santa Cruz Biotechnology (Dallas, USA).

Techniques: Luciferase, Activity Assay, Stable Transfection, Cell Culture, Staining, Transfection

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