goat anti-hdpp4 polyclonal antibody  (R&D Systems)

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Genome characteristics of pangolin MERS-like CoV, Manis javanica HKU4-related CoV (MjHKU4r-CoV) (A) Genome structure of MjHKU4r-CoV-1. (B) Similarity plot based on the full-length genome sequences. MjHKU4r-CoV-1 was used as a query sequence, Tylonycteris bat CoV HKU4-1, Pipistrellus bat CoV HKU5-1, bat MERS-related NeoCoV, and human MERS-CoV were used as reference sequences. (C) Phylogenetic tree based on the nucleotide sequences of the complete genomes of representative Alpha coronavirus (Alpha-CoV) and Beta coronavirus (Beta-CoV). MjHKU4r-CoV-1–4 are shown in red. Shaded colors represent different subgenera of coronaviruses. The scale bars represent 0.3 substitutions per nucleotide position. The software and setting used are described in the . (D) Schematic representation of the S protein. S1 and S2 subunits are indicated, as well as four domains within S1, including the N-terminal domain (NTD), RBD, subdomain 1 (SD1), and subdomain 2 (SD2). RBM alignment is shown at the bottom left, and the 16 key residues situated at the surface between MERS-CoV RBM and human DPP4 are pink shaded. Predicted furin cleavage sites of MjHKU4r-CoV-1 and the corresponding sites in other merbecoviruses are shown at the bottom right. The key arginine sites are shown in red, and the predicted furin cleavage sites are gray shaded. See also Figures S1 , , and and Table S1 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Sequencing, Software

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Isolation of MjHKU4r-CoV-1 and elucidation of its cell-entry mechanism (A) Transmission electron microscopy analysis of MjHKU4r-CoV-1 virions. Scale bars, 50 nm. (B) Cytopathic effect in Caco-2 cells at 48 h.p.i. with MjHKU4r-CoV-1 or mock infected (left, scale bars, 400 μm), and immunofluorescence assay (IFA) with an antibody against the Tylonycteris bat coronavirus HKU4 NP (right, scale bars, 200 μm). Red, NP; blue, nuclei. (C) Caco-2 cells were infected with MjHKU4r-CoV-1 at a multiplicity of infection (MOI) of 1 or 0.01. Viral titers and viral RNA copies at indicated time points were determined by TCID 50 assay and qRT-PCR, respectively. (D–G) Viral infection in Huh-7 cells with or without hDPP4 expression. Wild-type (WT) or DPP4 -knockout Huh-7 (KO) cells were infected with MjHKU4r-CoV-1 or MERS-CoV for 24 h. IFA was performed (D, scale bars, 200 μm) and quantified by high content screening (E), and viral titer (F) and RNA copy number (G) were determined in the supernatant. (H–K) Viral infection in KO cells after hDPP4 re-introduction. IF staining in Huh-7 KO cells transfected with hDPP4 expression plasmid or empty vector (H, scale bars, 200 μm) quantified by high content screening (I), and viral titer (J) and RNA copies (K) were determined in the supernatant at the indicated times. Data are presented as means and standard errors of the means (SEMs) of at least triplicate measurements in (E–G and I–K). Statistical significance was assessed using a two-tailed Student’s t -test in (E and I) and two-way ANOVA in (F, G, J, and K). See also Figure S4 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Isolation, Transmission Assay, Electron Microscopy, Infection, Immunofluorescence, Quantitative RT-PCR, Expressing, Knock-Out, High Content Screening, Staining, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 binds to human, bat, and pangolin DPP4 proteins (A) HeLa cells overexpressing the following DPP4 orthologous: hDPP4, Tylonycteris pachypus bat DPP4 ( Tp DPP4), Pipistrellus bat DPP4 ( Pp DPP4), or Manis javanica DPP4 ( Mj DPP4) were infected with MjHKU4r-CoV-1 at a MOI of 1 and subjected to IF staining at 24 h.p.i. Shown as MjHKU4r-CoV-1 NP or DPP4 protein expression (left, scale bars, 10 μm), or the replication dynamics of MjHKU4r-CoV-1 (viral titer, middle panel; genomic RNA, right panel). For IF staining, green, DPP4; red, NP; blue, nuclei. (B) Binding affinity of MjHKU4r-CoV-1, MERS-CoV, and HKU4-CoV RBD proteins to human, bat, and pangolin DPP4 proteins as measured by Bio-layer interferometry assay. The quantification of the binding affinity is shown in Table S2 . See also Figure S5 and Table S2 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Staining, Expressing, Binding Assay

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Human cell tropism and human colon ex vivo infection of MjHKU4r-CoV-1, related to Figure 5 (A) NP staining (red) of the indicated human cell lines at 24 h and 72 h.p.i. with MjHKU4r-CoV-1. Blue, nuclei; scale bars, 200 μm. Replication of MjHKU4r-CoV-1 in cell culture supernatant was analyzed by TCID 50 assay. (B–D) qRT-PCR detection of the replication of MjHKU4r-CoV-1 in human ex vivo colon tissue (B) or culture supernatant (C). IF staining of human ex vivo colon at 24 h.p.i. with MjHKU4r-CoV-1 (D). Red, viral NP; green, human DPP4; blue, nuclei; scale bars, 100 μm.

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Ex Vivo, Infection, Staining, Cell Culture, Quantitative RT-PCR

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 infection in human colon organoids and airway organoids (A–C) Replication of MjHKU4r-CoV-1 in human colon organoids (hCO), shown as viral copy numbers in cells (A) and supernatants (B) and viral titers in supernatants (C) as analyzed by qRT-PCR and TCID 50 assay, respectively. (D) Co-staining of MjHKU4r-CoV-1 NP (red) and hDPP4 (green) in human colon organoids at 72 h.p.i. (scale bars, 100 or 30 μm). Blue, nuclei. (E–G) Replication of MjHKU4r-CoV-1 in human airway organoids (hAWOs), shown as viral copy numbers in cells (E) and supernatants (F) and viral titers in supernatant (G) as analyzed by qRT-PCR and TCID 50 assay, respectively. (H) Co-staining of MjHKU4r-CoV-1 NP (red) and hDPP4 (green) in human airway organoids at 48 h.p.i. (scale bars, 50 or 10 μm). Blue, nuclei. See also Figure S6 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Quantitative RT-PCR, Staining

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 is infectious in hDPP4-Tg mice (A and B) hDPP4-Tg mice were intranasally inoculated with 1 × 10 6 TCID 50 MjHKU4r-CoV-1. (A) Mean body weights (n = 7 for MjHKU4r-CoV-1 infected mice and n = 3 for mock-infected mice), (B) body weights of individual mice. (C and D) qRT-PCR detection of viral replication in the lung (C) and brain (D) of MjHKU4r-CoV-1-infected hDPP4-Tg mice at the indicated time points (n = 4 for 1, 3, and 5 d.p.i. and n = 7 for 8 d.p.i.). (E) Lung and brain sections from MjHKU4r-CoV-1-infected or mock-infected mice stained for MjHKU4r-CoV-1 NP (red) at 5 d.p.i. (lung) and 8 d.p.i. (brain) (scale bars, 100 μm). Red, viral NP; blue, nuclei. (F) Pathological changes in the lungs of MjHKU4r-CoV-1-infected hDPP4-Tg mice at 5 d.p.i. Yellow arrows indicate severely affected areas (scale bars, 1,000 or 100 μm). See also Figure S7 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Quantitative RT-PCR, Staining

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet:

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Isolation, Recombinant, SYBR Green Assay, Luciferase, Reporter Assay, Sequencing, Knock-Out, Transgenic Assay, Plasmid Preparation, Software