monoclonal mouse igg 2a anti human antibody against ace2  (R&D Systems)

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Immunofluorescence and Western blot analysis of ACE2 in HEK-293 and HEK-293/ACE2 cell lines. (A,C) Immunofluorescence localization of ACE2 proteins (red fluorescence). (B,D) Immunostaining images with primary antibodies omitted (control). All images show cell nuclei stained with DAPI (blue fluorescence). The scale bar represents 20 µm. (E) Representative immunoblots for ACE2 (90 kDa) and Actin (42 kDa) as a loading control are shown for HEK-293 cells and the stable cell line overexpressing human ACE2 (HEK-293/ACE2). (F) The relative abundance of ACE2 protein levels is expressed as the ratio of ACE2 to Actin band intensities. Data are shown as mean ± SEM from three independent experiments.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Immunofluorescence, Western Blot, Fluorescence, Immunostaining, Control, Staining, Stable Transfection

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Effect of bromhexine on SARS-CoV-2 Omicron pseudovirus infectivity in HEK-293/ACE2 cells. (A,C,E) Representative fluorescence microscopy images of cells infected with Omicron pseudovirus treated with 1, 10, and 100 µM bromhexine, respectively. (B) Positive control (Omicron pseudovirus infection without bromhexine). (D) Negative control (cells without Omicron pseudovirus infection or bromhexine). (F) Quantitative analysis of Omicron pseudovirus infection in HEK-293/ACE2 cells, based on the percentage of GFP-positive cells after infection. GFP expression indicates successful pseudovirus entry. Data are presented as means ± SEM ( n = 4) from at least three different experiments. An asterisk (*) indicates statistically significant differences ( p < 0.05) compared to the positive control, determined by one-way ANOVA with post hoc Tukey HSD test.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Infection, Fluorescence, Microscopy, Positive Control, Negative Control, Expressing

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Luciferase activity and IC 50 determination on HEK-293/ACE2 cells. (A) Infectivity of Omicron pseudoviruses was assessed by measuring luciferase activity in relative luminescence units (RLUs) after infecting cells with the viruses. Data are shown as means ± SEM ( n = 4). An asterisk (*) indicates statistically significant differences ( p < 0.05) determined by one-way ANOVA with post hoc Tukey HSD test. (B) Dose-response curve used to determine the half-maximal inhibitory concentration (IC 50 ) of bromhexine in HEK-293/ACE2 cells infected with Omicron pseudovirus, with an IC 50 of 17.3 ± 0.9 µM. Results are presented as means ± SEM ( n = 4). Curves are fitted to a 4-parameter logistic model and generated using the average of fitted parameters from individual experiments.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Luciferase, Activity Assay, Infection, Concentration Assay, Generated

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Reduction in infectivity of SARS-CoV-2 pseudovirus variants in HEK-293/ACE2 cells treated with bromhexine. Infectivity of pseudoviruses representing Alpha, Beta, and Delta SARS-CoV-2 variants was measured by luciferase activity, expressed in relative luminescence units (RLUs), 48 h after treatment with 40 μM bromhexine or a vehicle (control). The cells were infected with pseudoviruses engineered to express each variant. Data are shown as means ± SEM ( n = 4). An asterisk (*) indicates statistically significant differences ( p < 0.05) compared to the control group, determined by two-way ANOVA followed by post hoc Tukey HSD test.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Infection, Luciferase, Activity Assay, Control, Variant 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: 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.)

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: 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.)

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: 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.

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: 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).

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

Journal: International journal of translational medicine (Basel, Switzerland)

Article Title: Organic Dust Exposure Enhances SARS-CoV-2 Entry in a PKC α - and ADAM-17-Dependent Manner

doi: 10.3390/ijtm4030032

Figure Lengend Snippet: ODE single and repetitive exposure increases murine lung soluble ACE levels dependent upon ADAM-17 with associated effect on SARS-CoV-2 pseudovirus infectivity. Scatter plots with bars depict mean with SEM of lung ACE2 levels of ( A ) wild-type (WT) mice following single and repetitive (13 times) ODE exposures and ( B ) humanized ACE2 mice treated with or without TAPI- 1, an ADAM-17 inhibitor, prior to single instillation with saline or 12.5% ODE exposure ( n = 6 mice/group). Humanized ACE2 mice were exposed to a single dose or repetitive doses of ODE prior to SARS-CoV-2 pseudovirus (PV) infection with lungs collected 5 days post-infection. ( C ) Scatter plot with mean and SEM depicted viral titer determined by qPCR ( n = 6–8 mice/group). * p < 0.05, ** p < 0.01, **** p < 0.0001; groups compared using Student’s t -test in ( A ) and one-way ANOVA with Tukey’s post hoc test in ( B , C ).

Article Snippet: Cells were washed and stained with monoclonal mouse IgG 2A anti-human antibody against ACE2 (Alexa Fluor 647, Clone 535919, R&D Systems) for 30 min on ice.

Techniques: Infection, Saline

Journal: International journal of translational medicine (Basel, Switzerland)

Article Title: Organic Dust Exposure Enhances SARS-CoV-2 Entry in a PKC α - and ADAM-17-Dependent Manner

doi: 10.3390/ijtm4030032

Figure Lengend Snippet: Inhibition of PKCα or ADAM-17 along with ODE treatment synergistically increases membrane ACE2 levels enhancing SARS-CoV-2 pseudovirus entry in BEAS-2B cells in vitro. ( A ) Wild-type (WT) and PKCα-deficient (DN) BEAS-2B cells were treated with Gö 6976 (a PKCα inhibitor), TAPI-1 (an ADAM-17 inhibitor), 0.5% ODE, or a combination for 1 h in vitro. The cells were then collected and stained for flow cytometry analysis of membrane ACE2 expression. ( B ) Treated cells were infected for 48 h with SARS-CoV-2 pseudovirus expressing fluorescent dTomato. The cells were then fixed, stained with Hoechst nuclear stain, and analyzed using Operetta CLS. WT cells treated with both ODE and inhibitor had significantly higher infection than single-treated WT groups. ( C ) Representative flow cytometry images showing ACE2 gating. ( D ) Representative immunofluorescence images from Operetta CLS showing pseudovirus-infected cells (20× magnification; scale bar: 100 μm). Data shown are mean ± SEM; n = 9 per group; experiments were repeated 4 times; ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA with Tukey’s post hoc test).

Article Snippet: Cells were washed and stained with monoclonal mouse IgG 2A anti-human antibody against ACE2 (Alexa Fluor 647, Clone 535919, R&D Systems) for 30 min on ice.

Techniques: Inhibition, Membrane, In Vitro, Staining, Flow Cytometry, Expressing, Infection, Immunofluorescence

Journal: International journal of translational medicine (Basel, Switzerland)

Article Title: Organic Dust Exposure Enhances SARS-CoV-2 Entry in a PKC α - and ADAM-17-Dependent Manner

doi: 10.3390/ijtm4030032

Figure Lengend Snippet: Proposed mechanism through which agricultural dust exposure could affect SARS-CoV-2 entry in vitro (created with BioRender.com ). Organic dust exposure (ODE) activates Toll-like receptor 2 (TLR2) and MyD88, which then activates protein kinase C alpha (PKCα). Through intermediates, PKCα activates ADAM-17 which cleaves the ACE2 receptor on the cell membrane producing soluble ACE2. PKCα can be inhibited by the addition of Gö 6976 and ADAM-17 can be inhibited by the addition of TAPI-1. If membrane ACE2 is intact, upon SARS-CoV-2 infection, the viral spike protein can bind the receptor and through unknown intermediates diminish IL-8 release in vitro.

Article Snippet: Cells were washed and stained with monoclonal mouse IgG 2A anti-human antibody against ACE2 (Alexa Fluor 647, Clone 535919, R&D Systems) for 30 min on ice.

Techniques: In Vitro, Membrane, Infection

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A-B) A549 cells were transduced to stably overexpress ACE2 (A549-A), DPP4 (A549-D) in the absence or presence of TMPRSS2 (A549-AT and A549-DT). ACE2 and DPP4 expression was confirmed by western blot (A) , while TMPRSS2 expression was confirmed using flow cytometry (B) . (C-D) A549-derived or Calu-3 cells were inoculated with lentiviral particles pseudotyped with the spike proteins of MERS-CoV (C) , VSV, SARS-CoV-1, WIV1-CoV, WIV16-CoV, SARS-CoV-2 Hu1, SARS-CoV-2 delta or SARS-CoV-2 omicron (D) or pseudoparticles lacking envelope protein (no env) (C-D) for 2 h, then incubated for an additional 72 h, at which point luciferase activity was measured to assess pseudoparticle entry. The data are expressed as fold change relative to the luciferase signal obtained with no envelope. Graphs show mean +/- SEM from three independent experiments performed in triplicate.

Article Snippet: For western blot, we used a rabbit monoclonal antibody against ACE2 (ThermoFisher #MA5-32307; diluted 1:1000), a rabbit monoclonal antibody against DPP4 (ThermoFisher #MA5-32643; diluted 1:1000), a rabbit monoclonal antibody against TMPRSS2 (Abcam #ab92323), and a mouse monoclonal antibody against GAPDH as a loading control (ThermoFisher #MA5-15738).

Techniques: Stable Transfection, Expressing, Western Blot, Flow Cytometry, Derivative Assay, Incubation, Luciferase, Activity Assay

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: BEAS-2B cells were transduced to stably overexpress ACE2 (BEAS-2B-ACE2). Expression of ACE2 and endogenous TMPRSS2 was confirmed by western blot (A). (B) BEAS-2B-ACE2 cells were pre-treated for 1 h at 37°C with DMSO, camostat (25 μM) or E64d (10 μM) diluted in media to the indicated concentrations, then infected with SARS-CoV-2pp for 2 h at 37°C. Inocula were removed and cells were incubated in complete media for 72 h, at which point luciferase activity was measured to assess viral entry. (C-D) BEAS-2B-ACE2 cells were pre-treated with NanH diluted to 50 μg/mL in serum-free media for 30 minutes at 37°C. Cells were then washed and processed for fluorescence microscopy (C) or inoculated with SARS-CoV-2 pseudoparticles (D) . NanH-treated cells were stained with SNA-FITC (binds sialic acid) or ECL-FITC (binds galactose) diluted to final concentration of 20 μg/mL in PBS, then washed with PBS and imaged by fluorescence microscopy (10X magnification; scale bar, 200 μm). Lectin staining confirmed removal of sialic acid by the NanH treatment. (E-F) BEAS-2B-ACE2 cells were treated with protease inhibitors or NanH as described (B-C) , then infected with replication-competent recombinant VSV-SARS-CoV-2-S expressing GFP for 2 h. After 7.5 h, cells were fixed and GFP fluorescence was assessed. Representative images are shown (20X magnification; scale bar, 50 μm). The percentage of infected cells in each condition was determined using ImageJ. Graphs show mean +/- SEM from three independent experiments performed in triplicate. Statistical significance was assessed by one-way or two-way ANOVA (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant).

Article Snippet: For western blot, we used a rabbit monoclonal antibody against ACE2 (ThermoFisher #MA5-32307; diluted 1:1000), a rabbit monoclonal antibody against DPP4 (ThermoFisher #MA5-32643; diluted 1:1000), a rabbit monoclonal antibody against TMPRSS2 (Abcam #ab92323), and a mouse monoclonal antibody against GAPDH as a loading control (ThermoFisher #MA5-15738).

Techniques: Stable Transfection, Expressing, Western Blot, Infection, Incubation, Luciferase, Activity Assay, Fluorescence, Microscopy, Staining, Concentration Assay, Recombinant

Journal: Cells

Article Title: Effects of Chronic Inflammatory Activation of Murine and Human Arterial Endothelial Cells at Normal Lipoprotein and Cholesterol Levels In Vivo and In Vitro

doi: 10.3390/cells13090773

Figure Lengend Snippet: ACE2 expression in human arterial endothelial cells upon inflammatory activation. Human arterial endothelial cells (HAECs) were treated for different periods of time with TNFα (50 ng/mL) or IL-1β (10 ng/mL), followed by RNA extraction. ( A ) RNA was reversed transcribed, and ACE2 mRNA was measured via quantitative PCR (mean ± SEM, n = 3, ANOVA was performed with GraphPad Prism 6.0 with Fisher’s LSD test between control and treated samples; p -values as indicated). ( B ) ACE2 protein levels were determined in HAECs treated as indicated by immunofluorescence staining and normalization of the staining intensity to the DNA staining (mean values ± SEM, n = 3; ANOVA and Fisher’s LSD test between treated samples and untreated control; * p < 0.05, ** p < 0.01, *** p < 0.0001).

Article Snippet: The cells were stained with primary rabbit monoclonal antibody against ACE2 (1:100, #MA5-32307, Invitrogen) and goat anti-rabbit Alexa Fluor 647 (1:500, #A21245, Invitrogen) antibody.

Techniques: Expressing, Activation Assay, RNA Extraction, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: Physiological Research

Article Title: Angiotensin I and II Stimulate Cell Invasion of SARS-CoV-2: Potential Mechanism via Inhibition of ACE2 Arm of RAS

doi: 10.33549/physiolres.935198

Figure Lengend Snippet: Characterization and use of HEK-ACE2 stable cell line. (a, b) Cytochemical and biochemical features of the model. The fluorescent photomicrographs of cells demonstrate the expression of ACE2 receptor on the cell surface (green: anti-ACE2 antibody, blue: nuclei stained with DAPI, scale bar: 20 μm) and Western blot analysis confirming the expression of ACE2. (c) Aa relative luminescence (RLU) recorded after incubation of HEK-ACE2 cells with PVPs for 48 h (MyBioSource) in serial dilutions (n=6). (d) The infectivity of PVPs produced in newly established cell line clones S/3 and S/7 in the cell-entry assay (n=5).

Article Snippet: To visualize the ACE2 expression in situ, the HEK-ACE2 cells were plated on cover glass pre-coated with rat-tail collagen, type I (Sigma-Aldrich), fixed with 4 % paraformaldehyde and treated with rabbit monoclonal antibody against human ACE2 at 10 μg/ml (Rockland Immunochemicals), followed by incubation with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Invitrogen).

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Incubation, Infection, Produced, Clone Assay

Journal: Physiological Research

Article Title: Angiotensin I and II Stimulate Cell Invasion of SARS-CoV-2: Potential Mechanism via Inhibition of ACE2 Arm of RAS

doi: 10.33549/physiolres.935198

Figure Lengend Snippet: The effect of Ang I and Ang II on PVP cell entry. The PVP infectivity (red line) was assessed using two cell lines (HEK-ACE2: a, b and Vero E6: c, d) at the indicated concentrations of peptides. The cell viability (blue line) was determined at the same concentrations of peptides in parallel cultures. Ang I and Ang II peptides stimulated the PVP entrance in HEK-ACE2 cells (red line, panel a, and b) at the concentration of 100 μM or higher (not shown), while the cell viability was not significantly decreased at the indicated concentrations (blue line). The increased infectivity of PVPs after the pre-treatment with Ang I (c) and Ang II (d) peptides in Vero E6 cells was observed; however, these cells showed decreased viability at the corresponding concentrations (blue line) (n=6, *** p<0.001; ** p<0.01; * p<0.05).

Article Snippet: To visualize the ACE2 expression in situ, the HEK-ACE2 cells were plated on cover glass pre-coated with rat-tail collagen, type I (Sigma-Aldrich), fixed with 4 % paraformaldehyde and treated with rabbit monoclonal antibody against human ACE2 at 10 μg/ml (Rockland Immunochemicals), followed by incubation with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Invitrogen).

Techniques: Infection, Concentration Assay

Journal: Physiological Research

Article Title: Angiotensin I and II Stimulate Cell Invasion of SARS-CoV-2: Potential Mechanism via Inhibition of ACE2 Arm of RAS

doi: 10.33549/physiolres.935198

Figure Lengend Snippet: In vitro metabolism of Ang I and Ang II in HEK-ACE2 and Vero E6 cells in absence and presence of PVP. (a) The expected products after the Ang I and Ang II cleavage by RAS enzymes. (b–e) Quantification of angiotensin peptides in cell culture (n=3, ** p<0.01; * p<0.05) as determined by liquid chromatography and mass spectrometry (Details are in Material and Methods).

Article Snippet: To visualize the ACE2 expression in situ, the HEK-ACE2 cells were plated on cover glass pre-coated with rat-tail collagen, type I (Sigma-Aldrich), fixed with 4 % paraformaldehyde and treated with rabbit monoclonal antibody against human ACE2 at 10 μg/ml (Rockland Immunochemicals), followed by incubation with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Invitrogen).

Techniques: In Vitro, Cell Culture, Liquid Chromatography, Mass Spectrometry

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

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.

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

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.

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).

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