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1.
Figure 5

Figure 5. From: Activation of innate immune antiviral response by NOD2.

Role of MAVS during NOD2-mediated activation of the antiviral pathway. (a) IFN-β production from mock and RSV infected primary normal human bronchial epithelial (NHBE) cells transfected with either control siRNA, NOD2 siRNA or MAVS siRNA. (b) IFN-β production from mock and RSV infected MEFs isolated from wild-type (WT), NOD2-KO or MAVS-KO mice. (c) IFN-β production from MEFs left untreated (UT) or stimulated with synthetic or viral (RSV) ssRNA. IFN-β was measured by ELISA and each value represents the mean ± s.d. from three independent experiments.

Ahmed Sabbah, et al. Nat Immunol. ;10(10):1073-1080.
2.
Figure 6

Figure 6. From: Activation of innate immune antiviral response by NOD2.

Interaction of MAVS with NOD2. (a) 293 cells were transfected with pcDNA, HA-NOD2 and-or GFP-MAVS and mock infected or infected with RSV. Lysates were immunoprecipitated with anti-HA agarose beads and bound proteins were immunoblotted with anti-GFP. Expression of GFP-IPS-1 in the cell lysate (by immunoblotting 25 µg of total cellular lysate with anti-GFP antibody) is also shown in the lower panel. (b) Expression of GFP-IPS-1 and HA-NOD2 in the cell lysate (by immunoblotting with anti-GFP and anti-HA antibodies) and amount of HA-NOD2 bound to anti-HA-agarose beads is also shown. For immunoblotting with cell lysates, 25 µg of total cellular lysate protein was used to detect GFP-IPS-1 and HA-NOD2. (c) RSV-infected (4h) 293 cells co-expressing GFP-MAVS (green) and HA-NOD2 (red) were imaged using confocal microscopy. (d,e) Mock or RSV-infected (6h) A549 cells (d) or RSV-infected (4h) NHBE cells (e) were stained with anti-NOD2 and anti-MAVS and imaged by confocal microscopy to detect endogenous NOD2 and MAVS.

Ahmed Sabbah, et al. Nat Immunol. ;10(10):1073-1080.
3.
Figure 3

Figure 3. From: Activation of innate immune antiviral response by NOD2.

NOD2 is required for IFN production. (a) RT-PCR analysis of NOD2 expression in mock and RSV infected A549 cells. (b) Activation of IRF3 luciferase reporter in mock and RSV infected (hours post-infection. p.i) A549 cells transfected with either control siRNA or NOD2 siRNA. The luciferase assay results are presented as mean ± s.d. from three independent experiments. (c) IFN-β production from mock and RSV infected primary normal human bronchial epithelial (NHBE) cells transfected with either control siRNA or NOD2 siRNA. (df) IFN-β production from mock and RSV infected alveolar macrophages (d), BMM (e) and MEFs (f) isolated from wild-type (WT) or NOD2-KO mice. IFN-β was measured by ELISA and each value represents the mean ± s.d. from three independent experiments.

Ahmed Sabbah, et al. Nat Immunol. ;10(10):1073-1080.
4.
Figure 1

Figure 1. From: Activation of innate immune antiviral response by NOD2.

ssRNA activates NOD2. (a) Activation of an IRF3 luciferase reporter in untreated (UT), ssRNA, and CpG DNA treated (6h) 293 cells transfected with pcDNA, human HA-NOD1 or human HA-NOD2. (b) RT-PCR analysis of NOD2 expression in A549 cells left untreated or stimulated for the indicated time periods with ssRNA. (c) Activation of IFN-β luciferase reporter in A549 cells transfected with either control siRNA or NOD2 siRNA and left untreated or stimulated (6h) with ssRNA. The luciferase assay results are presented as mean ± s.d. from three independent experiments. (d,e) Bone marrow-derived macrophages (BMM) or mouse embryonic fibroblasts (MEFs) were isolated from wild-type and NOD2-KO mice and left untreated or stimulated with ssRNA for the indicated time periods. IFN-β production was measured by ELISA. Values represent the mean ± s.d. of three independent experiments.

Ahmed Sabbah, et al. Nat Immunol. ;10(10):1073-1080.
5.
Figure 2

Figure 2. From: Activation of innate immune antiviral response by NOD2.

Activation of antiviral response by NOD2 in virus infected cells. (a,b) Activation of IRF3 and IFN-β luciferase reporter genes in mock infected and RSV infected 293 cells expressing pcDNA, HA-NOD1, or HA-NOD2. In (a), luciferase was measured 6 h post-infection (p.i.) and cells were infected with ultraviolet radiation (UV) treated or UV untreated RSV as indicated. (c) Plaque assay of VSV infectivity in 293 cells expressing pcDNA, HA-NOD1 or HA-NOD2. Crystal violet staining and VSV titer expressed as pfu per ml are shown. (d) RSV infectivity in 293 cells expressing pcDNA, HA-NOD1 or HA-NOD2. 100% infectivity represents the viral titer from cells expressing pcDNA. The plaque assay values represent the mean ± s.d. from three independent experiments. The luciferase assay results are presented as mean ± s.d. from three independent experiments.

Ahmed Sabbah, et al. Nat Immunol. ;10(10):1073-1080.
6.
Figure 7

Figure 7. From: Activation of innate immune antiviral response by NOD2.

NBD and LRR domains of NOD2 are essential for interaction with MAVS. (a) Schematic showing the various NOD2 constructs with deletion in specific domains. WT, wild-type; ΔCARD (NOD2 mutant lacking both CARD domains), ΔNBD (NOD2 mutant lacking the NBD domain) and ΔLRR (NOD2 mutant lacking the LRR domain). (b) 293 cells expressing various His-Myc tagged NOD2 constructs or pcDNA along with GFP- MAVS were lysed and lysates were incubated with Nickel-agarose (Ni-agarose). Following washing of the beads, the bound proteins were subjected to immunoblot analysis with anti-GFP. P; precipitation. (c) Lysates from cells in (b) were immunoblotted with anti-GFP to examine expression of GFP- MAVS. The first lane shows cells transfected with pcDNA only. (d) Cell lysates obtained from 293 cells co-expressing WT and NOD2 deletion mutants (his-myc-NOD2 constructs) along with GFP-IPS-1 was incubated with Ni-agarose, followed by immunoblotting with myc antibody (to detect his-myc tagged NOD2 protein constructs bound to the Ni-agarose beads). Please note that the WT and deleted version of NOD2 proteins are indicated with arrowheads.

Ahmed Sabbah, et al. Nat Immunol. ;10(10):1073-1080.
7.
Figure 8

Figure 8. From: Activation of innate immune antiviral response by NOD2.

NOD2 is essential for host defense against virus infection. (a) IFN-β concentrations in the bronchoalveolar lavage (BAL) of RSV infected wild-type (WT) and NOD2-KO mice. Values (n=four mice per group) are mean ± s.e.m. P < .05, by t-test when data were normally distributed, or by Mann-Whitney Rank sum test when data were not normally distributed. (b) RSV titer in the BAL (3d post-infection) of WT and NOD2-KO mice. Values are mean ± s.e.m. P < .05, by t-test when data normally distributed, or by Mann- Whitney Rank sum test when data were not normally distributed. (c) H&E staining of lung sections obtained from RSV-infected WT and NOD2-KO mice. (d) Neutrophil sequestration in lungs of RSV infected (2d post-infection) WT and NOD2-KO mice was assessed by myeloperoxidase (MPO) activity assay with total lung homogenate. MPO activity is shown as percentage increase over enzyme activity in mock infected mice. Results are mean ± s.e., n=5, P < 0.05. (e) TUNEL staining of lung sections obtained from RSV infected WT and NOD2-KO mice. (f) Survival of WT and NOD2-KO mice infected with RSV (5×108 pfu per animal). P › 0.02 between NOD2-KO and WT mice as deduced by Wilcoxon test.

Ahmed Sabbah, et al. Nat Immunol. ;10(10):1073-1080.
8.
Figure 4

Figure 4. From: Activation of innate immune antiviral response by NOD2.

Activation of NOD2 by viral ssRNA genome. (a) Activation of IRF3 luciferase reporter in 293 cells expressing pcDNA, HA-NOD1, HA-NOD2 that were left untreated (UT) or stimulated with RSV ssRNA genome (viral-ssRNA). Where indicated viral-ssRNA was treated with RNAse. The luciferase assay results are presented as mean ± s.d. from three independent experiments. (b,c) IFN-β production from BMM (b) and MEFs (c) isolated from wild-type (WT) or NOD2-KO mice that were left untreated or stimulated with viral-ssRNA. IFN-β was measured by ELISA and each value represents the mean ± s.d. from three independent experiments. (d) 293 cells were transfected with pcDNA or HA-NOD2 and were mock infected or infected with RSV. At 4h or 8h post-infection, NOD2 was immunoprecipitated with HA-agarose and bound RNA was amplified using primers specific for GAPDH or RSV nucleocapsid (N) protein. The amplified products were analyzed on the agarose gel. (e) RSV ssRNA genome and total cellular mRNA was incubated with HA-NOD2 bound to HA-agarose beads. Bound RNA was amplified using the primers in (d). The amplified products were analyzed on the agarose gel. Total cellular mRNA amplified with GAPDH specific primers served as a positive control.

Ahmed Sabbah, et al. Nat Immunol. ;10(10):1073-1080.

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