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