goat anti ngr1  (R&D Systems)

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: NgR1 is dispensable for reovirus replication and spread to the brain following peroral inoculation. (A) NgR1 expression in brain tissue lysates of WT or NgR1 -/- mice at postnatal days 4 (P4) and 10 (P10) were determined by immunoblotting using an antibody specific for mouse NgR1 (AF1440). (B-C) WT, JAM-A -/- , NgR1 -/- , and DKO mice were inoculated perorally with 10 4 PFU of reovirus T3SA-. Titers in the (B) intestine and (C) brain at the indicated intervals are shown. N = 8 to 9 animals per group for each time point. Error bars indicate SEM. Titers in tissues from knock-out animals were compared with WT at each time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; as determined by two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Expressing, Western Blot, Knock-Out

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: NgR1 is not required for reovirus neuropathogenesis following intracranial inoculation. (A-B) Newborn WT, JAM-A -/- , NgR1 -/- , and DKO mice were inoculated intracranially with 25 PFU of reovirus T3SA-. (A) Mice were monitored for disease signs and survival for 21 d and euthanized when moribund. N = 6 to 21 mice per group. Differences in survival in comparison to WT mice were not significant as determined by log-rank test. (B) At 3, 6, and 9 d after inoculation, mice were euthanized, brains were removed and hemisected, and viral titers in homogenates of the inoculated half of the brain were determined by plaque assay. Results are expressed as mean viral titers. Error bars indicate SEM. N = 8 to 11 mice per group for each time point. Differences in titer between WT and NgR1 -/- mice at each time point were not significant ( P > 0.05) as determined by two-way ANOVA with Dunnett’s multiple comparisons test. (C) Cortical neurons in culture isolated from E15.5 WT or NgR1 -/- mice were adsorbed with T3SA+ virions at an MOI of 500 PFU per cell. Cells were fixed at 24 h post-adsorption and immunostained using reovirus-specific antiserum. Infectivity was scored as the mean number of neurons identified based on morphology stained per field-of-view. Bars indicate means and error bars indicate SEM of triplicate samples from one representative experiment out of two conducted. **, P < 0.01; determined by t test.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Comparison, Plaque Assay, Isolation, Adsorption, Infection, Staining

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: Reovirus tropism in the brain is unaltered in the absence of NgR1. Newborn WT and NgR1 -/- mice were inoculated intracranially in the right brain hemisphere with 25 PFU of reovirus T3SA-. Mice were euthanized 6- or 9-d post-inoculation (dpi), and brains were removed and hemisected. Left-brain hemispheres were fixed in formalin and embedded in paraffin. Coronal sections of the left-brain hemisphere were stained with reovirus-specific antiserum and hematoxylin. Representative sections show reovirus antigen in cortex, hippocampus, thalamus, and cerebellum. Enlarged images of areas boxed in the overview show reovirus infection of neurons in the cortex and thalamus. Viral titers from the paired right-brain hemispheres are displayed adjacent to the micrographs.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Staining, Infection

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: NgR1 is dispensable for reovirus neural transmission following intramuscular inoculation. WT, JAM-A -/- , NgR1 -/- , and DKO mice were inoculated intramuscularly in the hindlimb with 5×10 6 PFU of reovirus T3SA-. Titers in the (A) inoculated hindlimb, (B) blood, (C) ISC, (D) SSC, and (E) brain at the indicated intervals are shown. Dotted lines indicate the limit of detection. N = 8 to 13 animals per group for each time point. Error bars indicate SEM. Titers in tissues from knock-out animals were compared with WT at each time point. *, P < 0.05; **, P < 0.01; ***; P < 0.001; as determined by two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Transmission Assay, Knock-Out

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: NgR1 is not required for replication of serotype 1 reovirus in the brain. Newborn WT and NgR1 -/- mice were inoculated intracranially with 100 PFU of reovirus T1L. At 3, 6, and 9 d after inoculation, mice were euthanized, brains were removed and hemisected, and viral titers in homogenates of the inoculated half of the brain were determined by plaque assay. Results are expressed as mean viral titers. Error bars indicate SEM. N = 7 to 8 mice per group for each time point. Differences in titer between WT and NgR1 -/- mice at each time point were not significant ( P > 0.05) as determined by two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Plaque Assay

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: The murine homolog of NgR1 does not efficiently engage reovirus. (A) CHO cells were transfected with plasmids encoding the proteins shown and incubated with 10 5 particles/cell of fluorescently-labeled reovirus T3SA- at 4°C for 1 h. The percentage of receptor-expressing cells bound by reovirus was quantified using flow cytometry. (B-C) Transfected CHO cells were adsorbed with 30 PFU/cell of T3SA-. (B) Infectivity was quantified at 24 h post-adsorption. Results are expressed as the mean viral values from duplicate samples of 2 independent experiments. Error bars indicate SEM. ns, P > 0.05; ****, P < 0.0001; as determined by one-way ANOVA with Dunnett’s multiple comparisons test. (C) Representative micrographs display nuclei stained with DAPI (blue), corresponding receptor expression (red), and reovirus antigen staining indicating infected cells (green). Scale bars, 200 μm.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Transfection, Incubation, Labeling, Expressing, Flow Cytometry, Infection, Adsorption, Staining

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: NgR1 is dispensable for reovirus replication and spread to the brain following peroral inoculation. (A) NgR1 expression in brain tissue lysates of WT or NgR1 -/- mice at postnatal days 4 (P4) and 10 (P10) were determined by immunoblotting using an antibody specific for mouse NgR1 (AF1440). (B-C) WT, JAM-A -/- , NgR1 -/- , and DKO mice were inoculated perorally with 10 4 PFU of reovirus T3SA-. Titers in the (B) intestine and (C) brain at the indicated intervals are shown. N = 8 to 9 animals per group for each time point. Error bars indicate SEM. Titers in tissues from knock-out animals were compared with WT at each time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; as determined by two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Expressing, Western Blot, Knock-Out

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: NgR1 is not required for reovirus neuropathogenesis following intracranial inoculation. (A-B) Newborn WT, JAM-A -/- , NgR1 -/- , and DKO mice were inoculated intracranially with 25 PFU of reovirus T3SA-. (A) Mice were monitored for disease signs and survival for 21 d and euthanized when moribund. N = 6 to 21 mice per group. Differences in survival in comparison to WT mice were not significant as determined by log-rank test. (B) At 3, 6, and 9 d after inoculation, mice were euthanized, brains were removed and hemisected, and viral titers in homogenates of the inoculated half of the brain were determined by plaque assay. Results are expressed as mean viral titers. Error bars indicate SEM. N = 8 to 11 mice per group for each time point. Differences in titer between WT and NgR1 -/- mice at each time point were not significant ( P > 0.05) as determined by two-way ANOVA with Dunnett’s multiple comparisons test. (C) Cortical neurons in culture isolated from E15.5 WT or NgR1 -/- mice were adsorbed with T3SA+ virions at an MOI of 500 PFU per cell. Cells were fixed at 24 h post-adsorption and immunostained using reovirus-specific antiserum. Infectivity was scored as the mean number of neurons identified based on morphology stained per field-of-view. Bars indicate means and error bars indicate SEM of triplicate samples from one representative experiment out of two conducted. **, P < 0.01; determined by t test.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Comparison, Plaque Assay, Isolation, Adsorption, Infection, Staining

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: Reovirus tropism in the brain is unaltered in the absence of NgR1. Newborn WT and NgR1 -/- mice were inoculated intracranially in the right brain hemisphere with 25 PFU of reovirus T3SA-. Mice were euthanized 6- or 9-d post-inoculation (dpi), and brains were removed and hemisected. Left-brain hemispheres were fixed in formalin and embedded in paraffin. Coronal sections of the left-brain hemisphere were stained with reovirus-specific antiserum and hematoxylin. Representative sections show reovirus antigen in cortex, hippocampus, thalamus, and cerebellum. Enlarged images of areas boxed in the overview show reovirus infection of neurons in the cortex and thalamus. Viral titers from the paired right-brain hemispheres are displayed adjacent to the micrographs.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Staining, Infection

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: NgR1 is dispensable for reovirus neural transmission following intramuscular inoculation. WT, JAM-A -/- , NgR1 -/- , and DKO mice were inoculated intramuscularly in the hindlimb with 5×10 6 PFU of reovirus T3SA-. Titers in the (A) inoculated hindlimb, (B) blood, (C) ISC, (D) SSC, and (E) brain at the indicated intervals are shown. Dotted lines indicate the limit of detection. N = 8 to 13 animals per group for each time point. Error bars indicate SEM. Titers in tissues from knock-out animals were compared with WT at each time point. *, P < 0.05; **, P < 0.01; ***; P < 0.001; as determined by two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Transmission Assay, Knock-Out

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: NgR1 is not required for replication of serotype 1 reovirus in the brain. Newborn WT and NgR1 -/- mice were inoculated intracranially with 100 PFU of reovirus T1L. At 3, 6, and 9 d after inoculation, mice were euthanized, brains were removed and hemisected, and viral titers in homogenates of the inoculated half of the brain were determined by plaque assay. Results are expressed as mean viral titers. Error bars indicate SEM. N = 7 to 8 mice per group for each time point. Differences in titer between WT and NgR1 -/- mice at each time point were not significant ( P > 0.05) as determined by two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Plaque Assay

Journal: bioRxiv

Article Title: The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

doi: 10.1101/2021.07.22.453368

Figure Lengend Snippet: The murine homolog of NgR1 does not efficiently engage reovirus. (A) CHO cells were transfected with plasmids encoding the proteins shown and incubated with 10 5 particles/cell of fluorescently-labeled reovirus T3SA- at 4°C for 1 h. The percentage of receptor-expressing cells bound by reovirus was quantified using flow cytometry. (B-C) Transfected CHO cells were adsorbed with 30 PFU/cell of T3SA-. (B) Infectivity was quantified at 24 h post-adsorption. Results are expressed as the mean viral values from duplicate samples of 2 independent experiments. Error bars indicate SEM. ns, P > 0.05; ****, P < 0.0001; as determined by one-way ANOVA with Dunnett’s multiple comparisons test. (C) Representative micrographs display nuclei stained with DAPI (blue), corresponding receptor expression (red), and reovirus antigen staining indicating infected cells (green). Scale bars, 200 μm.

Article Snippet: Cells were further incubated with the following primary antibodies to determine cell-surface expression of receptors: J10.4, mouse monoclonal antibody against JAM-A (provided by Charles Parkos, Emory University) ( ); rabbit monoclonal antibody against CAR (Sinobiological, 10799-R271); goat polyclonal antibody against human NgR1 (R&D Systems, AF1208); and goat polyclonal antibody against mouse NgR1 (R&D Systems, AF1440).

Techniques: Transfection, Incubation, Labeling, Expressing, Flow Cytometry, Infection, Adsorption, Staining