primary goat anti human ace2 antibody  (R&D Systems)

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Sequences of small-interfering RNAs

Article Snippet: BMEC were cultured to the logarithmic growth phase in 6-well plates (1 × 10 5 cells/well) and incubated for 12 h. Samples were then fixed in 4% formaldehyde for 15 min and permeated with 0.5% TritonX-100 for 10 min. Next, samples were blocked with 5% BSA for 1 h, incubated with anti-ACE2 primary antibody (1:500), followed by CY3-labeled fluorescent secondary antibody (1:1000, bs-0295G-Cy3, Goat anti-rabbit, Polyclonal, Bioss, Beijing, China) for 2 h. Finally, samples were washed three times with PBS followed by incubation with DAPI (100 ng/mL) for 5 min in the dark.

Techniques:

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Sequences of the primers used for RT-qPCR

Article Snippet: BMEC were cultured to the logarithmic growth phase in 6-well plates (1 × 10 5 cells/well) and incubated for 12 h. Samples were then fixed in 4% formaldehyde for 15 min and permeated with 0.5% TritonX-100 for 10 min. Next, samples were blocked with 5% BSA for 1 h, incubated with anti-ACE2 primary antibody (1:500), followed by CY3-labeled fluorescent secondary antibody (1:1000, bs-0295G-Cy3, Goat anti-rabbit, Polyclonal, Bioss, Beijing, China) for 2 h. Finally, samples were washed three times with PBS followed by incubation with DAPI (100 ng/mL) for 5 min in the dark.

Techniques: Sequencing

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Correlation analysis between LPS, inflammatory factors, and RAS pathway members. A Heat map showing the correlation between LPS, inflammatory cytokines and RAS members. Blue dots represent positive correlation and red dots represent negative correlation. Larger dots and darker colour correspond to greater correlation. Lower correlation is represented by smaller dots and lighter colour. B Ratio of ACE/ACE2 after treatment of BMEC with different concentrations of LPS. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: BMEC were cultured to the logarithmic growth phase in 6-well plates (1 × 10 5 cells/well) and incubated for 12 h. Samples were then fixed in 4% formaldehyde for 15 min and permeated with 0.5% TritonX-100 for 10 min. Next, samples were blocked with 5% BSA for 1 h, incubated with anti-ACE2 primary antibody (1:500), followed by CY3-labeled fluorescent secondary antibody (1:1000, bs-0295G-Cy3, Goat anti-rabbit, Polyclonal, Bioss, Beijing, China) for 2 h. Finally, samples were washed three times with PBS followed by incubation with DAPI (100 ng/mL) for 5 min in the dark.

Techniques:

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Distribution of ACE2 in BMEC. Cells were stained with antibodies against ACE2 and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Representative images were visualized by confocal laser microscopy. Red, ACE2; blue, DAPI. Scale bar: 10 μm.

Article Snippet: BMEC were cultured to the logarithmic growth phase in 6-well plates (1 × 10 5 cells/well) and incubated for 12 h. Samples were then fixed in 4% formaldehyde for 15 min and permeated with 0.5% TritonX-100 for 10 min. Next, samples were blocked with 5% BSA for 1 h, incubated with anti-ACE2 primary antibody (1:500), followed by CY3-labeled fluorescent secondary antibody (1:1000, bs-0295G-Cy3, Goat anti-rabbit, Polyclonal, Bioss, Beijing, China) for 2 h. Finally, samples were washed three times with PBS followed by incubation with DAPI (100 ng/mL) for 5 min in the dark.

Techniques: Staining, Microscopy

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: DAD3 induces ACE2 expression in LPS-induced BMEC. BMEC were treated with LPS (0.5 μg/mL), DA (20 μg/mL), DAD3 (20 μg/mL), LPS+DA (0.5 μg/mL+20 μg/mL) and LPS+DAD3 (0.5 μg/mL+20 μg/mL) for 24 h. A Relative ACE2 mRNA levels were determined by RT-qPCR. B ACE2 protein expression levels were determined by Western blotting. β-actin was used as a control. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: BMEC were cultured to the logarithmic growth phase in 6-well plates (1 × 10 5 cells/well) and incubated for 12 h. Samples were then fixed in 4% formaldehyde for 15 min and permeated with 0.5% TritonX-100 for 10 min. Next, samples were blocked with 5% BSA for 1 h, incubated with anti-ACE2 primary antibody (1:500), followed by CY3-labeled fluorescent secondary antibody (1:1000, bs-0295G-Cy3, Goat anti-rabbit, Polyclonal, Bioss, Beijing, China) for 2 h. Finally, samples were washed three times with PBS followed by incubation with DAPI (100 ng/mL) for 5 min in the dark.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DAD3 treatments on the transcriptional or expression levels of RAS members. A-E BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. The ACE2, AT1R and AT2R relative mRNA levels (transcriptional levels) of RAS members were determined by RT-qPCR. The Ang II and Ang-(1–7) protein expression levels of RAS members were determined by ELISA. A Relative ACE2 mRNA levels. B Expression levels of Ang II. C Expression levels of Ang-(1–7). D Relative AT1R mRNA levels. E Relative AT2R mRNA levels. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: BMEC were cultured to the logarithmic growth phase in 6-well plates (1 × 10 5 cells/well) and incubated for 12 h. Samples were then fixed in 4% formaldehyde for 15 min and permeated with 0.5% TritonX-100 for 10 min. Next, samples were blocked with 5% BSA for 1 h, incubated with anti-ACE2 primary antibody (1:500), followed by CY3-labeled fluorescent secondary antibody (1:1000, bs-0295G-Cy3, Goat anti-rabbit, Polyclonal, Bioss, Beijing, China) for 2 h. Finally, samples were washed three times with PBS followed by incubation with DAPI (100 ng/mL) for 5 min in the dark.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DAD3 treatments on the transcriptional levels of pro-inflammatory factors. A–D BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. Relative mRNA levels of pro-inflammatory factors were determined by RT-qPCR. A Relative IL-1β mRNA levels. B Relative IL-6 mRNA levels. C Relative IL-8 mRNA levels. D Relative TNF-α mRNA levels. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: BMEC were cultured to the logarithmic growth phase in 6-well plates (1 × 10 5 cells/well) and incubated for 12 h. Samples were then fixed in 4% formaldehyde for 15 min and permeated with 0.5% TritonX-100 for 10 min. Next, samples were blocked with 5% BSA for 1 h, incubated with anti-ACE2 primary antibody (1:500), followed by CY3-labeled fluorescent secondary antibody (1:1000, bs-0295G-Cy3, Goat anti-rabbit, Polyclonal, Bioss, Beijing, China) for 2 h. Finally, samples were washed three times with PBS followed by incubation with DAPI (100 ng/mL) for 5 min in the dark.

Techniques: Quantitative RT-PCR

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DA and DAD3 treatment on the expression levels of components of the MAPK and NF-κB pathways. A, B BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. The phosphorylation levels of p38, JNK1/2/3, ERK1/2 and IκB-α (p-p38, p-JNK, p-ERK1/2 and p-IκB-α) were determined by Western blotting. Activation of NF-κB pathway was indicated by p-IκB-α, an indicator for the activation of NF-κB. The band intensity of all detected proteins was normalized to β-actin and the expressions of p-p38, p-JNK, and p-ERK1/2 were normalized to p38, JNK, and ERK1/2, respectively. A DA treatments on the expression levels of components of the MAPK and NF-κB pathways; B DAD3 treatments on the expression levels of components of the MAPK and NF-κB pathways. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: BMEC were cultured to the logarithmic growth phase in 6-well plates (1 × 10 5 cells/well) and incubated for 12 h. Samples were then fixed in 4% formaldehyde for 15 min and permeated with 0.5% TritonX-100 for 10 min. Next, samples were blocked with 5% BSA for 1 h, incubated with anti-ACE2 primary antibody (1:500), followed by CY3-labeled fluorescent secondary antibody (1:1000, bs-0295G-Cy3, Goat anti-rabbit, Polyclonal, Bioss, Beijing, China) for 2 h. Finally, samples were washed three times with PBS followed by incubation with DAPI (100 ng/mL) for 5 min in the dark.

Techniques: Expressing, Western Blot, Activation Assay

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Immunohistochemistry confirms positive staining for ACE2 in several human tissue types. Representative fluorescent images of 4% formaldehyde fixed human tissue sections (n ≥ 3 independent donors, stained in duplicate) treated with ACE2 poly antibody (R&D AF933), visualised in green, and Hoechst 33342 nuclear stain, visualised in blue. Scale bars are as indicated in figure. Tissues shown include: ( a ) Kidney, with cortex and medulla indicated; ( b ) Kidney control section treated with secondary antibody and Hoechst 33342 alone; ( c ) Heart tissue, comprised predominantly of cardiomyocytes; ( d ) Lung, with preserved airway structures; ( e ) Liver, comprised predominantly of hepatocytes with preserved bile duct structures; ( f ) Hepatic artery section.

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Schematic showing the critical protein domains of full-length ACE2 versus the short dACE2 isoform. The 805 amino acid full-length ACE2 protein (left) is comprised of an extracellular domain that protrudes into the extracellular (E.C.) space and an intracellular domain that remains in the intracellular (I.C.) space. The extracellular domain is made up of a signal peptide (SP) that extends from positions 1–18; the peptide-binding catalytic site that covers 272–515; two spike protein binding sites (SB) located at 24–42 and 353–357; a collectrin-like domain (CLD) that covers 616–805; and a short transmembrane domain (TMD) that spans the membrane at positions 741–762. The short dACE2 isoform (right) loses all amino acids up to positon 357 and a unique 10 amino acid sequence caps the N-terminus. Note that dACE2 has lost both its spike protein binding sites and the catalytic site is non-functional. The diagram also shows the potential binding sites for the ACE2 poly antibody (green), raised against an 18–740 amino acid immunogen of ACE2, versus the single proprietary binding site that sits between amino acids 200–300 for the ACE2 mono antibody (orange). If the full ACE2 isoform is present, green and orange fluorescent signal will be observed in immunological staining studies. If only the short ACE2 isoform is present, green fluorescent signal alone will be observed due to the lack of the monoclonal antibody binding site. The schematic was generated using templates from Servier Medical Art (smart.servier.com).

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Binding Assay, Protein Binding, Sequencing, Functional Assay, Staining, Generated

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Differential expression of full-length ACE2 and the short dACE2 isoform in a panel of human tissues. Representative fluorescent images (n = 3 independent donors, stained in duplicate) of human tissue fixed in 4% formaldehyde and treated with ACE2 poly (left column, visualised in green) and ACE2 mono (centre column, visualised in orange). Merged images (right column, overlay) also include Hoechst 33342 nuclear marker (visualised in blue). Scale bars are as indicated in figure. Tissues shown include: ( a ) Kidney cortex, with a glomerulus (glom.) indicated; ( b ) Kidney border, showing a region where tubules of the cortex meet tubules of the medulla; ( c ) Heart tissue, showing cardiomyocytes; ( d ) Lung tissue, showing an airway structure; ( e ) Liver bile duct; ( f ) Liver hepatocytes.

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Expressing, Staining, Marker

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Quantification of differential expression of full-length ACE2 and the short dACE2 isoform in specific structures of human tissues. Graphical output shows the fold change in mean fluorescence observed for the respective secondary antibodies used to visualise ACE2 poly and ACE2 mono in distinct anatomical structures of the human tissue sections shown in Fig. . For each structure, n = 6 from ≥ 2 tissue sections. ( a ) Fold change in mean fluorescence observed for ACE2 poly and ACE2 mono in kidney cortex and glomeruli (Glom.), versus the normalised mean fluorescence observed in the renal medulla. ( b ) Fold change in mean fluorescence observed for ACE2 poly and ACE2 mono in airway structures and blood vessels (Blood ves.) of the lung, versus the normalised mean fluorescence observed in the connective tissue (Connec.). ( c ) Fold change in mean fluorescence observed for ACE2 poly and ACE2 mono in liver bile ducts and blood vessels (Blood ves.), versus the normalised mean fluorescence observed in liver hepatocytes (Hepato.). All data show mean ± SEM with individual data points shown. Statistical analyses of data included a one way ANOVA with multiple comparisons using Tukey’s correction. Statistical significance was determined where p < 0.05. **** or #### = p < 0.0001; ** = p < 0.01; n.s. = no significant difference.

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Binding of fluorescent SARS-CoV-2 spike-AF647 in ACE2 positive cells in a panel of human tissues. Representative confocal fluorescent images (n ≥ 2 independent donors, stained in duplicate) of human tissue fixed in 4% formaldehyde and treated with ACE2 poly (left column, visualised in green) and 1 µM spike-AF647 (centre column, visualised in red). Merged images (right column, overlay) also include Hoechst 33342 nuclear marker (visualised in blue). Scale bars are as indicated in figure. Tissues shown include: ( a ) Kidney cortex; ( b ) Kidney medulla; ( c ) Left ventricle heart tissue, showing cardiomyocytes; ( d ) Lung tissue, showing an airway structure; ( e ) Liver bile duct; ( f ) Liver hepatocytes.

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Binding Assay, Staining, Marker