Journal: Kidney international

Article Title: Chronic AT2 receptor activation attenuates renal AT1 receptor function and blood pressure in obese Zucker rats

doi: 10.1038/ki.2013.193

Figure Lengend Snippet: (A) ACE2 expression (B) ACE2 activity and (C) Mas receptor expression in the kidney cortex of control and CGP-treated lean and obese Zucker rats. Upper panels: Representative Western blots for respective proteins with loading control β-actin. For Western blot only, bar graphs represent the ratios of densities of respective protein bands and β-Actin i.e. ACE2/β-Actin, MasR/β-Actin $ significantly different compared with lean control rats, # significantly different compared with obese control rats. Values are represented as mean±SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05; N=5-7 in each group). (LCT- lean control, LCGP-lean treated with CGP42112A, OCT-obese control, OCGP-obese treated with CGP42112A).

Article Snippet: The expression of AT 1 R, AT 2 R, and renin, ACE, ACE2 and MasR in the idney cortex was determined by Western blotting and ACE2 activity was measured by Sensolyte 390 ACE2 activity kit (Anaspec Inc, CA) using spectrofluorometer (Cytofluor Series 4000, Applied Biosystem).For western blotting, the kidney cortices were homogenized in the buffer containing (in mM) Tris 50, EDTA 10, PMSF 1, cocktail of protease inhibitors (aprotinin, calpain inhibitors, leupeptin, pepstatin and trypsin inhibitor).

Techniques: Expressing, Activity Assay, Control, Western Blot

Journal: Kidney international

Article Title: Chronic AT2 receptor activation attenuates renal AT1 receptor function and blood pressure in obese Zucker rats

doi: 10.1038/ki.2013.193

Figure Lengend Snippet: Effect of AT 2 R agonist and antagonist on (A) ACE2 activity (B) MasR expression (C) Renin activity and (D) AT 1 R expression. Upper panels: Representative Western blots for respective proteins with loading control β-actin. Bar graphs: represent the ratios of densities of respective protein bands and β-Actin i.e AT 1 /β-Actin, Mas/β-Actin. *significantly different compared with control. Values are represented as mean ± SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05, N=6-7 in each group). (CT-control, CGP-CGP42112A, PD-PD123319).

Article Snippet: The expression of AT 1 R, AT 2 R, and renin, ACE, ACE2 and MasR in the idney cortex was determined by Western blotting and ACE2 activity was measured by Sensolyte 390 ACE2 activity kit (Anaspec Inc, CA) using spectrofluorometer (Cytofluor Series 4000, Applied Biosystem).For western blotting, the kidney cortices were homogenized in the buffer containing (in mM) Tris 50, EDTA 10, PMSF 1, cocktail of protease inhibitors (aprotinin, calpain inhibitors, leupeptin, pepstatin and trypsin inhibitor).

Techniques: Activity Assay, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) Schematic representation of our proposed treatments. SARS-CoV-2 infects ACE2 expressing cells (left panel). Binding of ACE2-Ig to SARS-CoV-2 Spike protein (middle panel) or binding of RBD-Ig to the ACE2 receptor (right panel) may prevent infection. (B) Staining of cells transfected to express ACE2 with an N-terminal Flag-tag (293T-ACE2 cells) and their parental cells that do not express a tag. This staining was performed using an anti-Flag antibody. (C) Staining of 293T-ACE2 cells with RBD-Ig. (D) Left panel: Spike protein surface expression on 293T cells co-transfected with either SARS-CoV-2 Spike envelope plasmid (293T-Spike cells) or Vesicular stomatitis virus (VSV) G envelope plasmid (293T-VSV-G cells). Right panel: Staining of 293T-Spike cells with ACE2-Ig. All histograms except from those made for 293T-Parental cells, were made from GFP positive gated cells. Figures shows one representative experiment out of 3 performed.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Expressing, Binding Assay, Infection, Staining, Transfection, FLAG-tag, Plasmid Preparation

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) ACE2 enzymatic activity assay. Recombinant human ACE2 and ACE2-Ig were incubated with and without an ACE2 inhibitor, then MCA based peptide substrate was added and plate was immediately inserted in the fluorescent plate reader. *p<0.005, **p<0.0005, ***p<0.00005, Student’s t-test as compared to same treatment with inhibitor. (B) Staining of 293T-Spike cells with ACE2-Ig which was previously incubated for 15 minutes with or without an ACE2 inhibitor. (C-D) Plaque reduction neutralization test. Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of either Control-Ig, ACE2-Ig (C) or RBD-Ig (D). % Neutralization was calculated as the percent of the decrease in plaque numbers, as compared with the background control. *P < 0.05; **P< 0.01; ***P <0.001; Student’s t-test as compared to Control-Ig. Figures shows one representative experiment out of 3 performed.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Enzyme Activity Assay, Recombinant, Incubation, Staining, Plaque Reduction Neutralization Test, Infection, Neutralization

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) Homozygous female K18-hACE2 transgenic mice were infected with SARS-CoV-2 (day 0) and treated with 75ug/mouse of either Control-Ig, RBD-Ig or ACE2-Ig. % of initial body weight was calculated from mice which were weighed daily. (B) Survival percentages of SARS-CoV-2 infected mice treated as described in A. *P < 0.05; Mantel-Cox test as compared to Infected + Control-Ig. Figure shows the combined results of two independent experiments.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Transgenic Assay, Infection

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) Anti-Spike IgG antibodies generated by mice following infection with SARS-CoV-2. Sera were taken 15 dpi from all mice groups and from naïve mice and diluted as indicated (upper right). Sera was incubated either with 293T-Spike cells (upper histograms) or with 293T-ACE2 cells (lower histograms) as a primary antibody then cells were stained with Alexa fluor 647 anti-mouse IgG secondary antibody. (B) ACE2-Ig staining of 293T-Spike cells in the presence or absence of sera from the various groups. Sera from all indicated groups were incubated with 293T-Spike cells for 1 hour at 4°C followed by staining with ACE2-Ig. All histograms were gated on GFP positive cells. Figure shows one representative experiment out of 2 performed.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Generated, Infection, Incubation, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) Staining of 293T-Parental cells and 293T-ACE2 cells with the mAb anti-ACE2 01 we generated. (B) Staining of 293T-Parental cells and 293T-ACE2 cells with RBD-Ig. Cells were incubated with or without anti-ACE2 01 for 1 hour at 4°C, washed and then staining was performed. (C) Staining of infected (MOI 0.5) and uninfected VERO E6 cells with either an anti-Spike antibody to verify infection (left panel) or with our anti-ACE2 01 antibody (right panel) at 16,24,48 hours PI. (D) Staining with anti-ACE 01 of 293T-ACE2 cells which were incubated with 1 ug of either Control-Ig or RBD-Ig for 1,2,6 and 24 hours. (A-D) All histograms were gated on GFP positive cells. (E) ACE2 enzymatic activity assay. Recombinant human ACE2 and 293T-ACE2 cells lysate (10 ug) were incubated with either Control-Ig (1 ug) or RBD-Ig (0.1 ug or 1ug), then MCA based peptide substrate was added and plate was immediately read in the fluorescent plate reader. Not significant (NS), Student’s t-test as compared with Control-Ig. (F) Plaque reduction neutralization test. Vero E6 cells were infected with increasing SARS-CoV-2 titers and treated with 20 ug/well of either Control-Ig, ACE2-Ig or RBD-Ig. % Neutralization was calculated as the percent of the decrease in plaque numbers, as compared with cells treated with Control-Ig. *P < 0.01; **P< 0.005; ***P <0.00001; Student’s t-test. Figures shows one representative experiment out of 3 (A-E) or 2 (F) performed.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Staining, Generated, Incubation, Infection, Enzyme Activity Assay, Recombinant, Plaque Reduction Neutralization Test, Neutralization