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1.
FIGURE 3.

FIGURE 3. From: Fas-associated Death Domain (FADD) and the E3 Ubiquitin-Protein Ligase TRIM21 Interact to Negatively Regulate Virus-induced Interferon Production.

Ubiquitination by TRIM21 is enhanced by FADD. A, 293T cells were transfected with HA-ubiquitin, FADD-FLAG, and TRIM21 in different combinations. FADD was immunoprecipitated from cell lysates using anti-FLAG antibodies, and immunoprecipitates were subsequently immunoblotted with anti-FLAG and anti-HA antibodies. B, His-ubiquitin, HA-TRIM21, and FADD-FLAG were transfected into 293T cells as indicated. Total cell extracts were examined by Western blot with anti-HA, anti-FLAG, and anti-β-actin antibodies. C, procedures were the same as B, except FADD siRNA was transfected where indicated (+).

Jennifer A. Young, et al. J Biol Chem. 2011 February 25;286(8):6521-6531.
2.
FIGURE 2.

FIGURE 2. From: Fas-associated Death Domain (FADD) and the E3 Ubiquitin-Protein Ligase TRIM21 Interact to Negatively Regulate Virus-induced Interferon Production.

Truncation and mutagenesis studies of FADD/TRIM21 interaction. A, the FADD-FLAG plasmid was co-transfected with wild-type HA-TRIM21 (wt), HA-TRIM21-(1–333), or HA-TRIM21-(131–473). Cell lysates were immunoprecipitated (IP) with anti-FLAG antibodies, and immunoprecipitates were Western-blotted with either HA- or FLAG-specific antibodies. The corresponding lysates were also Western-blotted as a control. B, the indicated plasmids were transfected into 293T followed by immunoprecipitation with FLAG-specific antibodies. The presence of FADD-associated TRIM21 was detected by anti-HA antibodies. C, the indicated FLAG-tagged FADD mutants were co-transfected with HA-TRIM21. FADD was immunoprecipitated with anti-FLAG antibodies, and associated TRIM21 was detected using HA-specific antibodies.

Jennifer A. Young, et al. J Biol Chem. 2011 February 25;286(8):6521-6531.
3.
FIGURE 5.

FIGURE 5. From: Fas-associated Death Domain (FADD) and the E3 Ubiquitin-Protein Ligase TRIM21 Interact to Negatively Regulate Virus-induced Interferon Production.

IRF7 associates with TRIM21 and FADD. A, 293T cells were transfected with the indicated expression plasmids. FADD was immunoprecipitated with anti-FLAG antibodies from uninfected or SeV-infected cell lysates. Immunoprecipitates were immunoblotted for IRF7, TRIM21, and FADD. B, 293T cells were transfected with IRF7 and FADD and TRIM21 where indicated. 24 h post-transfected, cells were stimulated with 50 HA units/ml SeV for 18–20 h. Whole cell lysates were immunoblotted to detect the levels of phosphorylated IRF7(*), total IRF7, TRIM21, FADD, and GAPDH. C, 293T cells were transfected with different combinations of HA-IRF7, ubiquitin, TRIM21, and FLAG-FADD. Cells were lysed, and IRF7 was immunoprecipitated with anti-HA antibodies. Immunoprecipitates were analyzed by Western blotting with anti-ubiquitin and anti-IRF7 antibodies. D, procedures were the same C, except cells were lysed under denaturing conditions and then diluted for anti-HA immunoprecipitations. For TRIM21 Western blot analysis, whole cell lysates (at 11% of the extracts used for immunoprecipitations (IP)) from cells transfected with ubiquitin, TRIM21, and FADD were loaded as a control. E, 293T cells were transfected with HA-IRF7, TRAF6-FLAG, FADD-FLAG, and TRIM21. Anti-HA antibodies were used to immunoprecipitate IRF7, and anti-ubiquitin antibodies were used to detect ubiquitinated IRF7.

Jennifer A. Young, et al. J Biol Chem. 2011 February 25;286(8):6521-6531.
4.
FIGURE 1.

FIGURE 1. From: Fas-associated Death Domain (FADD) and the E3 Ubiquitin-Protein Ligase TRIM21 Interact to Negatively Regulate Virus-induced Interferon Production.

FADD interacts with TRIM21 in vitro and in vivo. A, 293T cells were transfected with pCMV2-FLAG (vector) or pCMV2-FLAG-FADD (FADD). Cell were lysed and immunoprecipitated with anti-FLAG antibodies. The immunoprecipitates were incubated with additional extracts followed by six washes before running them on the gel. FADD and its co-immunoprecipitated proteins were visualized by silver staining and identified by the mass spectrometric analysis. B, HA-TRIM21, HA-TRIM39, HA-TRIM20, or an empty vector were transfected into 293T cells with (+) or without (−) FADD-FLAG. Anti-FLAG antibody was used to immunoprecipitate FADD, and the associating TRIM proteins were detected with an HA-specific antibody in a Western blot analysis. Total cell extracts were immunoblotted as a control for protein expression. C, 293T cell extracts were immunoprecipitated with rabbit anti-FADD or control antisera. Western blot analysis of the immunoprecipitates was then performed with anti-TRIM21 specific antibodies. D, cell extracts from 293T cells with or without Sendai virus infection were immunoprecipitated (IP) with anti-TRIM21 monoclonal antibodies or isotype control antibodies. Western blot analysis of the immunoprecipitates was then performed with anti-FADD or anti-TRIM21 specific antibodies.

Jennifer A. Young, et al. J Biol Chem. 2011 February 25;286(8):6521-6531.
5.
FIGURE 4.

FIGURE 4. From: Fas-associated Death Domain (FADD) and the E3 Ubiquitin-Protein Ligase TRIM21 Interact to Negatively Regulate Virus-induced Interferon Production.

Overexpression of FADD enhances IFN-β and NF-κB but attenuates IFN-α activation when co-transfected with TRIM21. 293T cells were transfected with an IFN-β-luciferase (A) or NF-κB-luciferase (B) reporter construct along with 0.3 μg of TRIM21 and/or FADD expression plasmids. Cells were stimulated with 50 HA units/ml SeV 24 h after transfection. Cells were lysed 18 h post-infections, and dual luciferase reporter assays (firefly and renilla) were performed. Samples were normalized with renilla luciferase. C, 293T cells were transfected with an IFNα4-luciferase reporter construct with IRF7 and 0.3 μg of TRIM21 and/or FADD expression plasmids. Cells were infected with SeV and lysed. Dual luciferase assays were performed as described in C. D, procedures were the same as C, except mutant FADD(D74A) was used where indicated along with 0.03 μg TRIM21 plasmid where indicated (0.03). All samples were normalized to renilla luciferase. All experiments were performed >3 times with similar results. E, left panel, 293T cells were transfected with TRIM21 and/or FADD, and expression of the endogenous IFN-β mRNA levels was measured by quantitative RT-PCR. Right panel, procedures were the same as the left panel, except cells were additionally transfected with IRF7, and IFN-α mRNA levels were measured. All experiments were performed three times with similar results.

Jennifer A. Young, et al. J Biol Chem. 2011 February 25;286(8):6521-6531.
6.
FIGURE 6.

FIGURE 6. From: Fas-associated Death Domain (FADD) and the E3 Ubiquitin-Protein Ligase TRIM21 Interact to Negatively Regulate Virus-induced Interferon Production.

Reduced TRIM21 or FADD expression leads to an increase in IFN-α activation and reduced viral replication. A, 293T cells were transfected with hTRIM21 siRNA, hFADD siRNA, or a control siRNA. Cells were lysed 48 h post-transfection, and extracts were immunoblotted with anti-TRIM21, anti-FADD, and anti-β-actin antibodies. B, left panel, an IFNα4-luciferase reporter construct was transfected into 293T cells with control siRNA, hTRIM21 siRNA, hFADD siRNA, or a combination of TRIM21 and FADD siRNAs with 50 ng of IRF7 expressing plasmid. After 24 h, cells were stimulated with 50 HA units/ml SeV for 18–20 h. Cells were then lysed and used in a dual luciferase reporter assay as previously described. Right panel, procedures were the same as the left panel, except IFN-α transcription was measured by quantitative RT-PCR. Left and right panel experiments were performed >3 and 2 times, respectively, with similar results. C, 293T cells were transfected with hTRIM21 siRNA, hFADD siRNA, or a control siRNA. Cells were stimulated with SeV similar to B, and IFN-β was measured by quantitative RT-PCR. D, procedures were similar to B, except extracts were blotted with HA-specific antibodies (for IRF7). E, TRIM21, FADD, or TRIM21/FADD knockdown experiments were done by the corresponding siRNAs. After confirmation of protein knockdown by Western blot analysis, cells were infected with influenza WSN at a multiplicity of infection of 1 for 1 or 2 days. Supernatants were harvested, and the TCID50/ml of the viruses in the supernatant were calculated by titration on Madin-Darby canine kidney cells. Experiments were performed 3 times with similar results.

Jennifer A. Young, et al. J Biol Chem. 2011 February 25;286(8):6521-6531.

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