Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 7

1.
FIGURE 5.

FIGURE 5. From: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response.

Role of IRF-5 in the antiviral and inflammatory response to NDV infection in vivo. IRF-5-deficient mice (Irf5/) and C57BL6/J (wt) mice were infected with NDV (50 HAU) for 6 h and then the collected serum was analyzed for type I IFN by bioassay (left), TNFα (middle), and IL-6 by ELISA (right). Data shown are combined from four independent experiments, wild type n = 12 and Irf5–/– n = 15 mice.

Andrea Paun, et al. J Biol Chem. 2008 May 23;283(21):14295-14308.
2.
FIGURE 1.

FIGURE 1. From: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response.

Characterization of mouse IRF-5 and its splice variants. A, protein sequence alignment of human IRF-5 variant 5 with mouse IRF-5 and the mouse IRF-5 BMv. The deletion in IRF-5 BMv is shown schematically both for the protein (B) and the genomic DNA sequences (C). L, molecular weight markers (ladder). Expression of ectopic MuIRF-5 (full-length (FL)) and IRF-5 BMv proteins in transfected 293T cells was identified by immune blotting (D), and mRNA expression was identified by RT-PCR (E). F, to detect the possible presence of IRF-5 splice variants in C57BL/6J (lane 1), BALB/c (lane 2), and NZB mice (lane 3), regions representing full-length IRF-5 and distinct exons were amplified by two-step RT-PCR using total splenic RNA.

Andrea Paun, et al. J Biol Chem. 2008 May 23;283(21):14295-14308.
3.
FIGURE 7.

FIGURE 7. From: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response.

The role of IRF-5 in the innate antiviral response. Following infection, viral nuclei acids are recognized by membrane-bound TLR and cytoplasmic RIG-I/MDA5 receptors. Our data, in conjunction with data of others (32), have shown that IRF-5 is activated by TLR7- or TLR9-, MyD88-dependent pathway. Activated IRF-5 stimulates expression of IFNA and IFNB genes as well as the inflammatory cytokines. However, although the role of IRF-5 in the activation of type I IFN genes is restricted to the MyD88 pathway and to cells that express the TLR7 or TLR9 receptors, the activation of the inflammatory genes by TLR4 and TLR3 seems also to be dependent on IRF-5, and consequently the role of IRF-5 in the induction of these inflammatory cytokines is broader and less cell type-restricted. There is no evidence that IRF-5 plays a substantial, if any, role in the RigI, IPS1 (VISA/MAVS/CARDif) pathway, and whether it can be activated by the MAD5 pathways is yet to be determined.

Andrea Paun, et al. J Biol Chem. 2008 May 23;283(21):14295-14308.
4.
FIGURE 2.

FIGURE 2. From: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response.

MuIRF-5 preferentially stimulates the murine IFNA4 gene promoter. A, 293T cells were transfected with the respective type I IFN promoter-luciferase reporter plasmids (50 ng), MuIRF-5 expressing plasmids (50 ng), or empty pcDNA6 vector and Renilla luciferase plasmid (10 ng). 20 h post-transfection cells were infected with NDV for 16 h and assayed for luciferase activity that was then normalized against Renilla luciferase. B, 293T cells were transfected with IRF-5 (50 ng) in the presence or absence of MyD88 (100 ng), with Renilla luciferase plasmid (10 ng) and type I IFN promoter-luciferase reporters (50 ng). C, 293T cells were transfected with IRF-5 full-length or IRF-5 BMv (100 ng) and MyD88 (50 ng) plasmids, IFNA4 reporter plasmid, and Renilla luciferase plasmid. Where indicated, cells were also infected with NDV for 16 h. D, 293T cells were transfected with IRF-5 full-length or IRF-5 BMv (10 ng), MyD88 (40 ng), PRDIII-I reporter plasmid, and Renilla luciferase plasmid. B and D, samples were assayed for luciferase activity 24 h post-transfection and normalized against Renilla luciferase. Data shown are combined from 2 or 3 independent experiments in A and C assays and triplicate repeats for B and D experiments.

Andrea Paun, et al. J Biol Chem. 2008 May 23;283(21):14295-14308.
5.
FIGURE 4.

FIGURE 4. From: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response.

Role of IRF-5 in TLR-dependent induction of inflammatory cytokines. 293T cells stably expressing TLR4 (A) or TLR2 (B) were transfected with luciferase reporters containing promoters of different inflammatory cytokines (40 ng), IRF-5 (100 ng), and Renilla luciferase (40 ng) plasmids. 8 h after transfection, cells were infected with Sendai virus (300 HAU/ml) or stimulated with Pam2Cys (10 nm) or LPS (10 ng/ml) in the presence of supernatants from MD2-expressing cells for 16 h. C, cells were transfected with the indicated amounts of IRF-5 and MyD88, Renilla luciferase (40 ng) plasmids, and TNFA reporter plasmid (40 ng) as described above. Transfected cells were left unstimulated or stimulated with 10 nm Pam2Cys for 16 h. D, 293T cells were transfected with IRF-5 (100 ng) and MyD88 or TBK1 (50 ng) plasmids, TNFA reporter, and Renilla luciferase plasmids. E, 293T cells were transfected as indicated with full-length IRF-5 or IRF-5 BMv (100 ng) and MyD88 (50 ng) plasmids, RANTES reporter plasmid, and Renilla luciferase plasmid. D and E samples were assayed for luciferase activity 24 h post-transfection and normalized against Renilla luciferase. Data shown are combined triplicates from two independent experiments.

Andrea Paun, et al. J Biol Chem. 2008 May 23;283(21):14295-14308.
6.
FIGURE 3.

FIGURE 3. From: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response.

MyD88-activated IRF-5 dimerizes and binds to the IFNA4 promoter. A, 293T cells were transfected with MyD88, full-length IRF-5, or IRF-5 BMv (all 0.5 μg) plasmids as indicated. Cells were lysed 24 h after transfection, proteins separated on a 7.5% native gel, and dimers detected by immunoblotting with anti-IRF-5 antibody. B, 293T cells were transfected as indicated with IRF-5 or IRF-3 and TBK1 plasmids (all 0.8 μg). 18 h after transfection cells were infected with NDV for 6 h. Proteins were analyzed on native and SDS-polyacrylamide gels and visualized by immunodetection. β-Actin levels were determined as a control for equal protein loading. C, in vivo binding of IRF-5 to the murine IFNA4 promoter was analyzed using the chromatin immunoprecipitation assay. The 293T cells were transfected with IFNA4 reporter (150 ng) and IRF-5 (110 ng) plasmids and either cotransfected with MyD88 plasmid (1.2 μg) or 18 h after transfection infected with NDV for 6 h. The chromatin immunoprecipitation assay was performed 24 h post-transfection as described under “Experimental Procedures.” Transfection with empty vector and immunoprecipitation with a nonspecific IgG antibody were used as controls. D, 293T cells (5 × 107) were transfected with a combination of IRF-3, IRF-5, MyD88, and TBK-1 plasmids, and 24 h after the transfection, cells were lysed under nondenaturing conditions, and IRF-5 was purified on Ni+-charged resin as described under supplemental Experimental Procedures. IB, immunoblot.

Andrea Paun, et al. J Biol Chem. 2008 May 23;283(21):14295-14308.
7.
FIGURE 6.

FIGURE 6. From: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response.

Cell type-specific role of IRF-5 in the antiviral and inflammatory response. A, total white blood cells from spleen and bone marrow cells (2 × 106 cells). B, peritoneal macrophages (2 × 106 cells) from WT and Irf5/ mice were infected with NDV (50 HAU) for 24 h. The levels of type I IFN in the culture medium were measured by bioassay. C, peritoneal macrophages from WT and Irf5/ mice were infected with NDV (50 HAU) and treated with R848 (10 μm) or LPS (20 ng/ml) for 24 h, and TNFα levels in the supernatant were measured by ELISA. D, BMDC. E, purified splenic CD11c+ DC (1 × 105 cells) from WT and Irf5/ mice were stimulated for 16 h with the respective TLR ligands, and levels of TNFα in the medium were measured by ELISA. F, purified splenic CD11c+ DC (1 × 106 cells) from WT and Irf5/ mice were stimulated for 2 h with respective TLR ligands. Cells were harvested; total RNA was isolated, and relative levels of IFNβ were measured by real time PCR. Transcript levels are shown in arbitrary units (A.U.) compared with β-actin.

Andrea Paun, et al. J Biol Chem. 2008 May 23;283(21):14295-14308.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk