rabbit anti-ace2 primary antibody  (Proteintech)

Journal: Advanced Science

Article Title: A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS‐CoV‐2 Spike‐Mediated Viral Invasion

doi: 10.1002/advs.202301478

Figure Lengend Snippet: Reconstitution of spike‐mediated viral invasion in the cell–vesicle content‐mixing assay. a) Schematic diagram of the ensemble content‐mixing assay between spike‐cell and ACE2‐vesicle. The spike‐cell and ACE2‐vesicle were mixed with 10 U 3C or 5 U Thr protease, and the fluorescence of dequenching signal of SRB was detected. b) Schematic illustration of the spike and its variants in which functional domains and cleavage sites are highlighted. In the mutants, the furin cleavage site (PRRAR) was mutated into the 3C cleavage site (LEVLFQGP), while the TMPRSS2 cleavage site (KPSKR) was mutated into the Thr cleavage site (LVPRGS). Meanwhile, the C‐terminal cytoplasmic domain of spike 1273 was deleted to obtain spike variants spike 1268 and spike 1234 . TM, transmembrane domain; CT, cytoplasmic tail. c) Western blot analysis of the cleavage state of spike 1273 on the surface of HEK293T cells, which was cleaved by the specified protease. Spike fragments were detected using anti‐RBD antibody targeting the N‐terminal spike and anti‐strep antibody targeting the C‐terminal spike. d) The ensemble content mixing of the spike 1273 ‐cell with ACE2‐vesicle. The fluorescence change of SRB for content mixing was normalized with respect to the fluorescence intensity obtained by adding 0.1% Triton X‐100. Traces represent the mean ± SEM from multiple repeats of independent experiments. e) Box plots and data points show maximum fusion probability corresponding to panel d. f) FCM shows the expression level of spike 1273 , spike 1268 and spike 1234 on the HEK293T cell surface. The monoclonal antibody 1A9 was used to target S2, and the expression signal from allophycocyanin (APC) that labeled the secondary antibody was detected. g) Box plots and data points show the percentage of spike‐positive cells corresponding to panel f. h) Western blot analysis of the expression of spike 1273 , spike 1268 and spike 1234 determined using anti‐RBD primary antibody. i) The ensemble content mixing of the spike‐cell with ACE2‐vesicle in the presence of Thr protease. Traces represent mean ± SEM from multiple repeats of independent experiments. j) Box plots and data points show the maximum fusion probability corresponding to panel i. In panels e, g and j, the repeats (N) of independent experiments were shown above the x‐axis, and statistical analysis was performed using two‐way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: For determining the expression level of ACE2, 1×10 6 cells were incubated with 1.07 µg mL −1 rabbit polyclonal anti‐ACE2 primary antibody (SinoBiological), followed by incubating with 1.88 µg mL −1 Alexa Fluor 488‐labeled goat anti‐rabbit IgG (H+L) secondary antibody, the others steps were the same as the aforementioned ones.

Techniques: Fluorescence, Functional Assay, Western Blot, Expressing, Labeling

Journal: Advanced Science

Article Title: A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS‐CoV‐2 Spike‐Mediated Viral Invasion

doi: 10.1002/advs.202301478

Figure Lengend Snippet: RBD binding to ACE2 is critical for spike‐mediated membrane fusion. a) The interface of the RBD and ACE2 protein (PDB:6LZG) shown in the ribbon diagram, in which RBD and ACE2 are colored in cyan and purple respectively. The key residues that involved in the interface of the RBD and ACE2 were labeled. b) Schematic diagram of the spike 1268 and mutations on the RBD domain are shown. c) The ensemble content mixing of the spike 1268 or its mutants‐expressing HEK293T cells with ACE2‐vesicle in the presence of 5 U Thr protease. Traces represent mean ± SEM from multiple repeats of independent experiments. d) Box plots and data points show the maximum fusion probability corresponding to panel c. e) Schematic diagram of ACE2. The mutations on ACE2 are shown as red. SP, signal peptide; PD, peptidase domain; ND, neck domain; TM, transmembrane domain; CT, cytoplasmic tail. f) The ensemble content mixing of the spike 1268 ‐cell with vesicles reconstituted with ACE2 or its mutants in the presence of 5 U Thr protease. Traces represent mean ± SEM from multiple repeats of independent experiments. g) Box plots and data points show the maximum fusion probability corresponding to panel f. h) The ensemble content mixing of the spike 1268 ‐cell with ACE2‐vesicle at different molar ratios of ACE2: lipids in the presence of 5 U Thr protease. Traces represent mean ± SEM from multiple repeats of independent experiments. i) Box plots and data points show the maximum fusion probability corresponding to panel h. In panels d, g, and i, the repeats (N) of independent experiments were shown above the x‐axis, and statistical analysis was performed using two‐way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: For determining the expression level of ACE2, 1×10 6 cells were incubated with 1.07 µg mL −1 rabbit polyclonal anti‐ACE2 primary antibody (SinoBiological), followed by incubating with 1.88 µg mL −1 Alexa Fluor 488‐labeled goat anti‐rabbit IgG (H+L) secondary antibody, the others steps were the same as the aforementioned ones.

Techniques: Binding Assay, Membrane, Labeling, Expressing

Journal: Advanced Science

Article Title: A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS‐CoV‐2 Spike‐Mediated Viral Invasion

doi: 10.1002/advs.202301478

Figure Lengend Snippet: Spike drives membrane fusion by crosslinking two membranes in the cell–vesicle content‐mixing assay. a) Schematic diagram showing the in vitro cell–vesicle content‐mixing assay between spike 1268 ‐cell and blank‐vesicle by crosslinking membranes via the NTV_biotin interaction. b) The ensemble cell–vesicle content mixing of the spike 1268 ‐cell with ACE2‐vesicle or blank‐vesicle. Traces represent mean ± SEM from multiple repeats of independent experiments in the presence of 5 U Thr protease. c) Box plots and data points show the maximum fusion probability corresponding to panel b. d) The ensemble cell–vesicle content‐mixing assay of the spike 1268 ‐cell with blank‐vesicle at specified concentration of NTV in the presence of 5 U Thr protease. Traces represent mean ± SEM from multiple repeats of independent experiments e) Box plots and data points show the maximum fusion probability corresponding to panel d. In Panels c and e, the repeats (N) of independent experiments were shown above the x‐axis, and statistical analysis was performed using two‐way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: For determining the expression level of ACE2, 1×10 6 cells were incubated with 1.07 µg mL −1 rabbit polyclonal anti‐ACE2 primary antibody (SinoBiological), followed by incubating with 1.88 µg mL −1 Alexa Fluor 488‐labeled goat anti‐rabbit IgG (H+L) secondary antibody, the others steps were the same as the aforementioned ones.

Techniques: Membrane, In Vitro, Concentration Assay

Journal: Advanced Science

Article Title: A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS‐CoV‐2 Spike‐Mediated Viral Invasion

doi: 10.1002/advs.202301478

Figure Lengend Snippet: Spike induces syncytia formation by crosslinking two cell membranes. a) Schematic diagram of the cell–cell fusion assay in the presence of ACE2 (upper), or NTV (bottom). b) FCM shows the expression level of ACE2 on the ACE2‐cell and uninfected‐cell. The rabbit polyclonal antibody was used to target ACE2, and the signal of Alexa Fluor 488‐labeled the secondary antibody was detected. c) Box plots and data points show the percentage of ACE2‐positive cells corresponding to panel b. d) Syncytia formation induced by spike 1268 mediated cell–cell fusion via the NTV_biotin interaction or the RBD_ACE2 interaction. After spike 1268 ‐EGFP‐cell, ACE2‐cell, and uninfected‐cell were treated with biotin‐PE, spike 1268 ‐EGFP‐cell and ACE2‐cell (or uninfected‐cell) were co‐cultured at a 2:1 ratio with or without 5 U Thr protease. Scale bar, 200 µ m . e) Box plots and data points show the area of syncytia in cell–cell fusion corresponding to panel d. The area of syncytia was calculated based on the fused cells with weak fluorescence intensity of EGFP and were at least twice as large as the unfused cells. At least five fields were randomly selected in each well. Statistical analysis was performed using two‐way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: For determining the expression level of ACE2, 1×10 6 cells were incubated with 1.07 µg mL −1 rabbit polyclonal anti‐ACE2 primary antibody (SinoBiological), followed by incubating with 1.88 µg mL −1 Alexa Fluor 488‐labeled goat anti‐rabbit IgG (H+L) secondary antibody, the others steps were the same as the aforementioned ones.

Techniques: Cell-Cell Fusion Assay, Expressing, Labeling, Cell Culture, Fluorescence

Journal: Advanced Science

Article Title: A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS‐CoV‐2 Spike‐Mediated Viral Invasion

doi: 10.1002/advs.202301478

Figure Lengend Snippet: Spike induces syncytia formation at an optimal distance of cell membranes. a) Schematic diagram of the cell–cell fusion assay in the presence of ACE2 (upper), or lipid‐anchored complementary DNA strands (bottom). Cholesterol‐linked DNA‐A was reconstituted onto spike 1268 ‐EGFP‐cell, while cholesterol‐linked DNA‐D was reconstituted onto uninfected‐cell. b) Syncytia formation was induced by spike 1268 mediated cell–cell fusion. Spike 1268 ‐EGFP‐cell containing DNA‐A was co‐cultured with uninfected‐cell containing specified DNA‐D at a 2:1 ratio. Scale bar, 200 µ m . c) Quantitative analysis of the area of syncytia in cell–cell fusion shown in b, statistical analysis was performed using two‐way ANOVA among the experiments in the presence of Thr, and the statistical analysis within dashed line was compared to the experiment without DNA strands followed by Tukey's multiple comparisons test.

Article Snippet: For determining the expression level of ACE2, 1×10 6 cells were incubated with 1.07 µg mL −1 rabbit polyclonal anti‐ACE2 primary antibody (SinoBiological), followed by incubating with 1.88 µg mL −1 Alexa Fluor 488‐labeled goat anti‐rabbit IgG (H+L) secondary antibody, the others steps were the same as the aforementioned ones.

Techniques: Cell-Cell Fusion Assay, Cell Culture

Journal: Advanced Science

Article Title: A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS‐CoV‐2 Spike‐Mediated Viral Invasion

doi: 10.1002/advs.202301478

Figure Lengend Snippet: Spike‐mediated membrane fusion can be inhibited by manipulating the distance between two membranes. a) Schematic diagram showing the inhibition of spike mediated membrane fusion between spike 1268 ‐cell and ACE2‐vesicle by altering the distance between two membranes. A pair of cholesterol linked complementary DNA (long DNA pair [129 bp]; short DNA pair [6 bp]) or PE‐linked PEG5000 was reconstituted onto spike 1268 ‐cell and ACE2‐vesicle. b) The ensemble cell–vesicle content‐mixing assay of the spike 1268 ‐cell with ACE2‐vesicle in the presence of specified membrane crosslinker in the presence of 5 U Thr protease. c) Box plots and data points show the maximum fusion probability corresponding to panel b. d) Spike mediated viral invasion initiated by the distance regulator of the RBD_ACE2 interaction. Viral invasion is induced by the formation of six‐helix bundle of HR1 and HR2, following the action of cleavage at the S2’ site. e) Spike mediated viral invasion initiated by other distance regulators. In the absence of ACE2, spike alone does not drive viral invasion. However, when membrane crosslinker bridges two membranes at a suitable distance, spike is capable of driving viral invasion in the absence of ACE2. Statistical analysis was performed using two‐way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: For determining the expression level of ACE2, 1×10 6 cells were incubated with 1.07 µg mL −1 rabbit polyclonal anti‐ACE2 primary antibody (SinoBiological), followed by incubating with 1.88 µg mL −1 Alexa Fluor 488‐labeled goat anti‐rabbit IgG (H+L) secondary antibody, the others steps were the same as the aforementioned ones.

Techniques: Membrane, Inhibition

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: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 1. DHA reduces both ACE1 and ACE2 levels in key rat tissues. Western blotting was used to measure ACE1 and ACE2 levels relative to total protein load, as

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 2. DHA differentially modulates ACE1 and ACE2 levels in growing and quiescent EA.hy926 en- dothelial cells. Western blotting was used to compare ACE1 and ACE2 relative to total protein load, as measured by Ponceau S in growing cells treated with ALA, EPA, or DHA at the indicated concentrations (µM) for (a) 8 and (b) 24 h, as well as in quiescent cells treated with n3-PUFA for (c) 8 and (d) 24 h. The band intensities of ACE2 and ACE1 were quantified and are graphically presented in panels (e,f), respectively; the 100 kDa band (grey bars) represents non-glycosylated ACE2, whereas the 130 kDa band (black bars) corresponds to N-glycosylated ACE2. Data are presented as mean ± SEM, n = 3; bars not sharing a common letter in the graphs are significantly different (p < 0.05) based on Duncan’s multiple range or LSD post-hoc tests.

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 5. Schematic of proposed mechanism of action. LCn3-PUFAs, EPA and DHA, reduce ACE2 protein levels. These n3-PUFAs may also decrease ACE2 glycosylation. These mechanisms may explain how DHA blocks SARS-CoV-2 pseudovirus entry into HEK293 cells. LCn3-PUFAs also downregulate ACE1 protein levels, thus maintaining the balance between ACE1 and ACE2, and diminishing the risk of adverse CVD outcomes. This figure was prepared by S. Huang using Microsoft PowerPoint software version 16.16.27.

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Glycoproteomics, Software