anti human ace2 polyclonal goat igg  (ATCC)

Journal: Frontiers in cellular and infection microbiology

Article Title: Carbohydrate-binding protein from stinging nettle as fusion inhibitor for SARS-CoV-2 variants of concern.

doi: 10.3389/fcimb.2022.989534

Figure Lengend Snippet: FIGURE 1 Antiviral activity of UDA against pseudotyped SARS-CoV-2. VLPs expressing the Wuhan-Hu-1 spike and a GFP or luciferase reporter was used to transduce cells in the absence (untreated control) or presence of UDA (as indicated). (A) A549.ACE2+ cells were transiently transfected with TMPRSS2 and subsequently exposed to pseudotyped SARS-CoV-2 expressing GFP. Panel shows representative images taken at 3 days post- transduction. (B) GFP-positive cells were quantified from images of (A). Graph represents a concentration-response of UDA (mean ± SD from 3 independent experiments). (C) UDA was tested against luciferase-based pseudotyped SARS-CoV-2 in commercially available A549.ACE2+.TMPRSS2+

Article Snippet: The following antibodies were used for western blotting: ACE2 Polyclonal Goat IgG (cat. n° AF933, R&D systems), antib-actin (cat. n° MA1-140, Invitrogen), HRP-labelled goat antimouse immunoglobulin (IgG; cat. n° P0447, Dako).

Techniques: Activity Assay, Expressing, Luciferase, Transduction, Control, Transfection, Concentration Assay

Journal: Frontiers in cellular and infection microbiology

Article Title: Carbohydrate-binding protein from stinging nettle as fusion inhibitor for SARS-CoV-2 variants of concern.

doi: 10.3389/fcimb.2022.989534

Figure Lengend Snippet: FIGURE 2 Antiviral activity of UDA against live SARS-CoV-2 virus in Vero E6 and U87.ACE2+ cells. Cells were exposed to clinical isolates of SARS-CoV-2, i.e., variants Wuhan-Hu-1 (A), 20A.EU2 (B), Gamma (C) or GFP-expressing Wuhan-Hu-1 (D) in the absence or presence of UDA. (A, B) SARS- CoV-2 replication was assessed by RT-qPCR analysis of the viral copy numbers of the N gene in the supernatant at day 3 post infection (p.i.). RT-qPCR data were used to calculate the % inhibition of viral replication and to plot a concentration-response curve for UDA. Graphs show data of 3 independent experiments with 2 technical replicates each (mean ± SD; n=6). (C) Cells were collected at 40h p.i. and stained intracellularly for the viral N protein. Histogram plots show mean fluorescence intensity (MFI) values of N expression in noninfected (Cell Control; grey), infected (Virus Control; blue) and UDA-treated infected (red) Vero E6 cells from a representative experiment. Single cell analysis was performed on 8,000 – 10,000 cells by flow cytometry. The numbers in each plot refer to the percentage of cells that stained positive for N (i.e., infected cells). The dashed grey histogram plot represents the background signal from the non-infected cell control. (D) Pictures, taken at 2 days post infection, show GFP expression in the infected U87.ACE2+ cells. Representative pictures from a biological replicate out of two are shown. The values between brackets refer to the percentage GFP+ area (relative to the virus control); mean ± SD (n=2).

Article Snippet: The following antibodies were used for western blotting: ACE2 Polyclonal Goat IgG (cat. n° AF933, R&D systems), antib-actin (cat. n° MA1-140, Invitrogen), HRP-labelled goat antimouse immunoglobulin (IgG; cat. n° P0447, Dako).

Techniques: Activity Assay, Virus, Expressing, Quantitative RT-PCR, Infection, Inhibition, Concentration Assay, Staining, Control, Single-cell Analysis, Cytometry

Journal: Frontiers in cellular and infection microbiology

Article Title: Carbohydrate-binding protein from stinging nettle as fusion inhibitor for SARS-CoV-2 variants of concern.

doi: 10.3389/fcimb.2022.989534

Figure Lengend Snippet: FIGURE 3 Antiviral activity of UDA against live SARS-CoV-2 virus in A549 cells and primary ALI cultures. (A, B) A549.ACE2+-TMPRSS2 cells were exposed to SARS-CoV-2 (WT Wuhan-Hu-1) in the absence or presence of UDA. (A) SARS-CoV-2 replication was assessed by flow cytometry. Cells infected with SARS-CoV-2 in the absence or presence of UDA (2 µM) were collected at 48h p.i. and stained intracellularly for the viral N protein. Histogram plots show mean fluorescence intensity (MFI) values of N expression in noninfected (Cell Control; grey), infected (Virus Control; blue) and UDA-treated infected (red) cells from a representative experiment. Single cell analysis was performed on 8,000 – 10,000 cells by flow cytometry. The numbers in each plot refer to the percentage of cells that stained positive for N (i.e., infected cells). The dashed grey histogram plot represents the background signal from the non-infected cell control. (B) SARS-CoV-2 replication was assessed by RT-qPCR analysis of the viral copy numbers of the N gene in the supernatant at day 3 post infection (p.i.). RT-qPCR data were used to calculate the % inhibition of viral replication and to plot a concentration-response curve for UDA. Graphs show data of 3 independent experiments with 2 technical replicates each (mean ± SD; n=6). (C) Human primary lower (SmallAir) airway epithelial ALI cultures were infected apically with a GFP-expressing SARS- CoV-2 variant (Wuhan-Hu-1) for 2h in the absence or presence of UDA, washed and exposed to air. Pictures, taken at 4 days post infection, show GFP expression in the infected cells. Representative pictures from a biological replicate out of two are shown.

Article Snippet: The following antibodies were used for western blotting: ACE2 Polyclonal Goat IgG (cat. n° AF933, R&D systems), antib-actin (cat. n° MA1-140, Invitrogen), HRP-labelled goat antimouse immunoglobulin (IgG; cat. n° P0447, Dako).

Techniques: Activity Assay, Virus, Cytometry, Infection, Staining, Expressing, Control, Single-cell Analysis, Quantitative RT-PCR, Inhibition, Concentration Assay, Variant Assay

Journal: Frontiers in cellular and infection microbiology

Article Title: Carbohydrate-binding protein from stinging nettle as fusion inhibitor for SARS-CoV-2 variants of concern.

doi: 10.3389/fcimb.2022.989534

Figure Lengend Snippet: FIGURE 4 Surface plasmon resonance (SPR) analysis of UDA. (A) SPR sensorgram showing the binding kinetics for UDA and immobilized monomeric Wuhan-Hu-1 spike protein (1:2 dilutions of UDA, starting from 100 nM). Data are shown as black lines, and the best fit of the data to a 1:1 binding model is shown in red. (B) SPR sensorgram showing the binding kinetics for UDA and immobilized RBD of Wuhan-Hu-1 spike protein (1:2 dilutions of UDA, starting from 200 nM), with a fast off rate. Data are shown as black lines, and the best fit of the data to a 1:1 binding model is shown in red. (C) Biotinylated ACE2 was coupled as ligand to a CAP sensor chip. Graph shows sensorgrams for the binding of different analytes to ACE2. Green curve: RBD (50 nM) only; red curve: RBD (50 nM) + UDA (1 µM); grey curve: RBD (50 nM) + non-neutralising spike- binding antibody R007 (50 nM); blue curve: RBD (50 nM) + spike-neutralising antibody R001 (50 nM); orange curve: UDA (1 µM) only. Note that RBD in complex with R007 can still bind to ACE2 resulting in a stronger resonance signal induced by the large protein complex. See Supplementary Figure 4A for kinetics values.

Article Snippet: The following antibodies were used for western blotting: ACE2 Polyclonal Goat IgG (cat. n° AF933, R&D systems), antib-actin (cat. n° MA1-140, Invitrogen), HRP-labelled goat antimouse immunoglobulin (IgG; cat. n° P0447, Dako).

Techniques: SPR Assay, Binding Assay

Journal: Frontiers in cellular and infection microbiology

Article Title: Carbohydrate-binding protein from stinging nettle as fusion inhibitor for SARS-CoV-2 variants of concern.

doi: 10.3389/fcimb.2022.989534

Figure Lengend Snippet: FIGURE 5 UDA prevents cell-cell fusion of A549.ACE2+ cells with spike-expressing HEK293T cells. A549.ACE2+ cells (transfected to express the first 10 betasheets of neongreen) were overlayed with HEK293T cells co-transfected with a plasmid encoding the SARS-CoV-2 spike protein and a plasmid encoding the 11th betasheet of neongreen. Overlay was done in the absence (untreated control) or presence of UDA. Representative pictures of cell-cell fusion were taken 12h after the co-cultivation of both cell types. Top row (‘no pretreatment’): compound was added at the same moment as cell overlay; Second row (‘HEK293T pretreated; wash’): HEK293T were pretreated with UDA for 30 min; extensively washed and added to the A549 cells without compound; Third row (‘HEK293T pretreated’): HEK293T were pretreated with UDA for 30 min and added to the A549 cells with compound; Fourth row (‘A549.ACE2+ pretreated; wash’): A549 cells were pretreated with UDA for 30 min; extensively washed before the HEK293T cells were added without compound; Fifth row (‘A549.ACE2+ pretreated’): A549 cells were pretreated with UDA for 30 min before HEK293T cells were added without removal of compound; Bottom row (‘HEK293T non-transfected’): as a negative control HEK293T cells were transfected with only the 11th betasheet of neongreen (without spike protein) and were added to the A549.ACE2+ cells (transfected with the first 10 betasheets of neongreen). For each condition, 2 replicate wells were analysed and in each well 4 different areas of the cell culture were monitored using an Incucyte live-cell analysis instrument. Representative pictures are shown. Neongreen expression analysis of the pictures is summarized in Supplementary Figure 5B.

Article Snippet: The following antibodies were used for western blotting: ACE2 Polyclonal Goat IgG (cat. n° AF933, R&D systems), antib-actin (cat. n° MA1-140, Invitrogen), HRP-labelled goat antimouse immunoglobulin (IgG; cat. n° P0447, Dako).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Negative Control, Cell Culture, Cell Analysis