anti irf7 ab  (Bio-Rad)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 4. RelB deficiency triggers elevated basal expressions of T1-IFNs involving IRF7-mediated positive autoregulation. (A) GSEA comparing WT and Relb−/−

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 5. T1-IFNs and IRF7 cooperatively restrict viral multiplication in Relb−/−cells. Plaque assay reveal- ing the titer of progeny virus particles produced by WT and Relb−/−cells, including primary MEFs (A) or BMDMs (C, left panel) or immortalized MEFs (C, right panel), upon infection for 12 h with the indicated MOI of CHPV. (B) RT-qPCR showing the relative abundance of viral genomic RNA and mRNAs encoding viral nucleo- capsid protein and phosphoprotein in WT and Relb−/−

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques: Plaque Assay, Virus, Produced, Infection, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 6. Vitamin D modulates the T1-IFNIRF7 pathway through RelB to exert antiviral effects. Vitamin D naive or vitamin Dconditioned WT or Relb−/−primary MEFs were examined for the relative abundance of IRF7 mRNA (A) and protein (B). Similarly, vitamin Dinduced expressions of IFN-b mRNA were scored in Relb−/−MEFs (C) and immortalized IRF7DCRISPR MEFs (E). Virus propaga- tion was scored in vitamin D naive and vitamin Dconditioned Relb−/−MEFs (D) or immortalized IRF7DCRISPR MEFs (F) subsequent to cell infection with 0.1 or 2 MOI of CHPV. The RT-qPCR data represent mean ± SEM of three indepen- dent experiments. The immunoblots represent three experi- mental replicates. The plaque assay data represent mean ± SEM of four biological replicates. *p# 0.05, **p# 0.01.

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques: Virus, Infection, Quantitative RT-PCR, Western Blot, Plaque Assay

Journal: Frontiers in Immunology

Article Title: Grass Carp ( Ctenopharyngodon idella ) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7

doi: 10.3389/fimmu.2021.808159

Figure Lengend Snippet: Sequences and applications of primers used in this study.

Article Snippet: Anti-IRF7 Ab (1:1000) was from HUABIO Biological and used as previously described ( ).

Techniques: Sequencing, Cloning, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Grass Carp ( Ctenopharyngodon idella ) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7

doi: 10.3389/fimmu.2021.808159

Figure Lengend Snippet: Ci KAT8 blocks IRF3/IRF7-induced IFN 1 expression. CO cells seeded in (6-well or 24-well) plates were single-transfected or co-transfected with 2 µg of pcDNA3.1-KAT8 and pcDNA3.1-IRF3/IRF7. pcDNA3.1-basic was used as a control group. 36 h later, cells were harvested for detection of the mRNA level of IFN 1 (A) or luciferase activities of IFN 1 promoter and ISRE element (B) , or the protein level of IFN 1 (C) . * ( p < 0.05) and ** ( p < 0.01). Data are representative of three independent experiments (C) . The histograms exhibit the relative expression levels, which are quantified using ImageJ software.

Article Snippet: Anti-IRF7 Ab (1:1000) was from HUABIO Biological and used as previously described ( ).

Techniques: Expressing, Transfection, Control, Luciferase, Software

Journal: Frontiers in Immunology

Article Title: Grass Carp ( Ctenopharyngodon idella ) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7

doi: 10.3389/fimmu.2021.808159

Figure Lengend Snippet: Ci KAT8 interacts with IRF3 and IRF7 via the MYST domain. (A–D) CO cells seeded in 60mm dishes were co-transfected with 1.5 µg of KAT8-Flag and 1.5 µg of (IRF3-GFP or IRF7-GFP) plasmids, 36 h later, cell lysates were immunoprecipitated with anti-Flag Ab (A, C) or anti-GFP Ab (B, D) , then Western blot analyzed the interaction of KAT8 with IRF3/7. (E, F) CO cells seeded in 60 mm dishes were transfected with 1.5 µg KAT8-Flag or Flag-tagged mutant KAT8 (△1-151, △151-264, △264-487) plus (IRF3-GFP or IRF7-GFP), 36 h later, cell lysates were immunoprecipitated with anti-Flag Ab, Western blot analyzed the interaction of three KAT8 truncations with IRF3/IRF7. “*” refers to IRF3-GFP or IRF7-GFP. The molecular masses of KAT8-Flag, KAT8-(△1-151)-Flag, KAT8-(△151-264)-Flag, KAT8-(△264-487)-Flag, IRF3-GFP, IRF7-GFP are 60, 42, 46, 34, 85, and 82 kD, respectively. Data are representative of three independent experiments.

Article Snippet: Anti-IRF7 Ab (1:1000) was from HUABIO Biological and used as previously described ( ).

Techniques: Transfection, Immunoprecipitation, Western Blot, Mutagenesis

Journal: Frontiers in Immunology

Article Title: Grass Carp ( Ctenopharyngodon idella ) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7

doi: 10.3389/fimmu.2021.808159

Figure Lengend Snippet: Ci KAT8 acetylates IRF3/7 via its MYST domain. (A, B) CO cells seeded in 60 mm dishes were co-transfected with 1.5 µg of cmv-Flag or KAT8-Flag and 1.5 µg of IRF3-GFP plasmids (A) , or 1.5 µg of cmv-Flag or KAT8-Flag and IRF7-GFP plasmids (B) . 36 h later, the cell lysates were added with the deacetylase inhibitors cocktail and then co-immunoprecipitated with an anti-GFP antibody. Eventually, Western blot was performed with the indicated antibodies. (C, D) CO cells were transfected with control siRNA (N.C) or specific siRNA targeting KAT8 (siKAT8) for 12 h, then transfected with IRF3-GFP (C) or IRF7-GFP (D) plasmids for other 24 h, respectively. The subsequent experiment was conducted similarly to (A, B, E, F) Effect of KAT8 and its truncated mutants on the acetylation of IRF3/IRF7, CO cells were transfected with the indicated plasmids for 36 h, and the subsequent experiment was conducted similarly to A, (B) The molecular masses of KAT8-Flag, KAT8-(△1-151)-Flag, KAT8-(△151-264)-Flag, KAT8-(△264-487)-Flag, IRF3-GFP, IRF7-GFP are 60, 42, 46, 34, 85, and 82 kD, respectively. (G–I) CO cells seeded in plates (6-well or 24-well) were transfected with 2 µg of pcDNA3.1-KAT8-WT or pcDNA3.1-KAT8-(△1-151) or pcDNA3.1-KAT8-(△151-264) or pcDNA3.1-KAT8-(△264-487). 36 h later, cells were harvested for detection of the mRNA level of IFN 1 (G) or the protein level of IFN 1 (H) or the luciferase activities of IFN 1 and ISG15 promoters (I) * (p < 0.05) and ** (p < 0.01). Data are representative of three independent experiments (A–F, H) . The histograms show relative expression levels quantified using the Image J software. "ns" means no significance.

Article Snippet: Anti-IRF7 Ab (1:1000) was from HUABIO Biological and used as previously described ( ).

Techniques: Transfection, Histone Deacetylase Assay, Immunoprecipitation, Western Blot, Control, Luciferase, Expressing, Software

Journal: Frontiers in Immunology

Article Title: Grass Carp ( Ctenopharyngodon idella ) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7

doi: 10.3389/fimmu.2021.808159

Figure Lengend Snippet: Ci KAT8 inhibits the combination of IRF3/IRF7 with ISRE response element. (A, B) CO cells seeded in 60 mm dishes and transfected with 1.5 µg of cmv-FLAG or KAT8-FLAG puls 1.5 µg of IRF3-GFP or IRF7-GFP. 36 h later, the cell extracts were incubated with immobilized biotin-labeled ISRE or No-biotin labeled ISRE. 12 h later, the beads were washed with lysis buffer three times, eluted with 2 × SDS sample buffer and boiled at 95°C for 10 min. Western blot was used to detect the affinity of IRF3/IRF7 with ISRE response element. Data are representative of three independent experiments.

Article Snippet: Anti-IRF7 Ab (1:1000) was from HUABIO Biological and used as previously described ( ).

Techniques: Transfection, Incubation, Labeling, Lysis, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection.

doi: 10.4049/jimmunol.2001186

Figure Lengend Snippet: FIGURE 2. PEDV M protein significantly inhibited type I IFN and ISGs expression. (A) HEK-293T cells were transfected with 2 mg of plasmids express- ing M protein or empty vector. At 24 hpt, the cells were infected with SeV for 16 h. The mRNA expression levels of IFN-b, IFN-a1, IFN-a2, ISG15, ISG54, and ISG56 were determined and analyzed by qPCR. (B) HEK-293T cells were cotransfected with 0.5 mg of HA-IRF7expressing plasmid and 1.5 mg of M-expressing plasmids or empty vector. At 24 hpt, the mRNA expression levels of IFN-a1 and IFN-a2 were analyzed by qPCR. (C) PK-15 cells were transfected with poly (I:C) for 12 h, and the cells were then infected or mock-infected with PEDV at an MOI of 1 for 18 h. The mRNA expression levels of IFN-b, ISG15, and ISG54 were measured by qPCR. (D) PK-15 cells were transfected with IRF7 (2 mg) for 24 h, and the cells were then infected or mock-in- fected with PEDV at an MOI of 1 for another 18 h. The mRNA expression levels of IFN-a1 and IFN-a2 were detected by qPCR. (E) PK-15 cells were trans- fected with 3 mg of HA-tagged M-expressing plasmid or empty vector. At 24 hpt, the cells were infected with SeV for 18 h. The mRNA expression levels of IFN-b, ISG15, ISG54, and ISG56 were examined by qPCR. (F) PK-15 cells were cotransfected with 1 mg of HA-IRF7expressing plasmid and 2.5 mg of plasmids expressing M protein or empty vector. At 24 hpt, the mRNA expression levels of IFN-a1 and IFN-a2 were determined by qPCR. Experiments were performed in triplicate and repeated three times. Graphs showed mean ± SD. Asterisks indicate statistical significance calculated by the Student t test. *p < 0.05, **p < 0.01.

Article Snippet: For virus infection assays, Vero cells were infected with PEDV (multiplicity of infection [MOI] of 1) at 37 C for 18 h. The PEDV-infected or mock-infected cells were prepared as described above and the primary Abs were Rabbit antiPEDV M and mouse anti-IRF7 Ab (Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection.

doi: 10.4049/jimmunol.2001186

Figure Lengend Snippet: FIGURE 3. M proteins of both epidemic and vaccine strains inhibited type I IFN production and ISGs expression. (A) Alignment of the M protein amino acid sequences of PEDV CV777 strain and the field strains GDS07, B5-HB2017, and CH-HNKF-16. The asterisk marked the different amino acid among these strains (the fifth amino acid). (B) M proteins of both epidemic and vaccine strains inhibited IFN-b promoter activation. HEK-293T cells were cotrans- fected with the pIFN-bLuc reporter plasmid (0.05 mg), pRL-TK plasmid (0.005 mg), and the plasmids expressing M protein, M (F5S) protein, or empty vec- tor. At 24 hpt, the cells were infected with SeV. The luciferase activities were measured at 16 hpi. The expression of M protein and M (F5S) was confirmed by Western blotting analysis. (C) HEK-293T cells were transfected with 2 mg of plasmids expressing M protein, M (F5S) protein, or empty vector. At 24 hpt, the cells were infected with SeV for 16 h. The mRNA expression levels of IFN-b, ISG15, and ISG54 were measured by qPCR. (D) HEK-293T cells were transfected with 0.5 mg of IRF7, together with 1.5 mg of plasmids expressing M protein, M (F5S) protein, or empty vector. At 24 hpt, the mRNA ex- pression levels of IFN-a1 and IFN-a2 were measured by qPCR. (E) PK-15 cells were transfected with 1 mg of HA-IRF7expressing plasmid, together with 2.5 mg of plasmids expressing M protein, M (F5S) protein, or empty vector. At 24 hpt, the mRNA expression levels of IFN-a1 and IFN-a2 were measured by qPCR. (F) PK-15 cells were transfected with IRF7 (2 mg) for 24 h, and the cells were then infected or mock-infected with epidemic PEDV strain at an MOI of 1 for another 36 h. The mRNA expression levels of IFN-a1 and IFN-a2 were detected by qPCR. Three independent experiments were carried out in triplicate with similar results. Results were representative of the three independent experiments. Graphs showed mean ± SD. The statistical significance was calculated by the Student t test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Article Snippet: For virus infection assays, Vero cells were infected with PEDV (multiplicity of infection [MOI] of 1) at 37 C for 18 h. The PEDV-infected or mock-infected cells were prepared as described above and the primary Abs were Rabbit antiPEDV M and mouse anti-IRF7 Ab (Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Plasmid Preparation, Infection, Luciferase, Western Blot, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection.

doi: 10.4049/jimmunol.2001186

Figure Lengend Snippet: FIGURE 4. PEDV M protein inhibited RIG-I signaling by targeting IRF3, IRF7, or the downstream molecules. (A) HEK-293T cells were cotransfected with pcDNA3.1-M-HA (0.1 mg), pIFN-bLuc (0.05 mg), and pRL-TK (0.005 mg) along with a series of constructs expressing RIG-I-CARD, VISA, TBK1, IRF3, or IRF7 (0.05 mg). Dual-luciferase assays were performed at 24 hpt. The expression of PEDV M protein was confirmed by Western blotting. Results were representative of three independent experiments performed in triplicates. Graphs show mean ± SD. The statistical significance was calculated by the Student t test. (B) M protein did not inhibit the protein expression of various components of RIG-I signal pathway (RIG-I, VISA, TANK, IKKe, TBK1, IRF3, and IRF7). HEK-293T cells were cotransfected with vector plasmids or pcDNA3.1-M-HA plasmids with the constructs expressing various components of RIG-I pathway. The cells were collected at 36 hpt, and the whole-cell lysates were analyzed by Western blotting with the indicated Abs. The experiments were repeated three times with similar results. Data were representative of the three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Article Snippet: For virus infection assays, Vero cells were infected with PEDV (multiplicity of infection [MOI] of 1) at 37 C for 18 h. The PEDV-infected or mock-infected cells were prepared as described above and the primary Abs were Rabbit antiPEDV M and mouse anti-IRF7 Ab (Santa Cruz Biotechnology).

Techniques: Construct, Expressing, Luciferase, Western Blot, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection.

doi: 10.4049/jimmunol.2001186

Figure Lengend Snippet: FIGURE 5. PEDV M protein interacted with IRF7. (A) HEK-293T cells were cotransfected with pcDNA3.1-M-myc and the indicated HA-tagged adaptor molecule expressing plasmids. The cells were collected at 36 hpt, and the cell lysates were immunoprecipitated by anti-myc Ab (IP: myc). The whole-cell lysates (WCLs) and immunoprecipitation (IP) complexes were analyzed by Western blotting with the indicated Abs. (B) HEK-293T cells were cotransfected with HA- IRF7 and pcDNA3.1 empty vector plasmid or pcDNA3.1-M-mycexpressing plasmids. The cells were then collected at 36 hpt, and the cell lysates were immu- noprecipitated with control IgG or anti-myc Ab (IP: myc). The WCLs and IP complexes were analyzed by Western blotting with the indicated Abs. (C) HEK- 293T cells were cotransfected with pcDNA3.1 empty vector or pcDNA3.1-M-HA and Flag-tagged IRF7-expressing plasmids. The cells were lysed at 36 hpt and then immunoprecipitated with control IgG or anti-Flag Ab (IP: Flag). The WCLs and IP complexes were analyzed by Western blotting using the indicated Abs. (D and E) HEK-293T cells were transfected with pcDNA3.1-M-HA or empty vector. The cell lysates were immunoprecipitated with control IgG or anti-IRF7 (D) or anti-HA (E) Ab. The WCLs and IP complexes were analyzed by Western blotting using the indicated Abs. (F) HEK-293T cells were transfected with pcDNA3.1-M-HA, pCMV-Flag-nsp10 or empty vector plasmids. The expression of IRF7, M-HA, and Flag-nsp10 was detected by indirect immunofluorescent assay (IFA) analysis at 24 hpt. The cells were double immunostained for IRF7 (red) and M-HA (green) or Flag-nsp10 (green); cellular nuclei were counter- stained with DAPI (blue). Original magnification 130 (Leica TCS SP5 II AOBS). (G) HEK-293T cells were transfected with pcDNA3.1-M-HA or empty vec- tor. The cells were lysed at 36 hpt and the expression of IRF7 and M was detected by Western blotting using the indicated Abs. (H) Vero cells were mock- infected or infected with PEDV (MOI of 1) for 18 h. The expression of IRF7 and M protein was detected by IFA analysis with anti-IRF7 and antiPEDV M Abs. Original magnification 63 (Leica TCS SP5 II AOBS). The Pearson correlation coefficient was analyzed using the Image-Pro Plus 6.0 software. (I) Sche- matic representation of a series of IRF7 mutants. (J) HEK-293T cells were cotransfected with Flag-IRF7expressing plasmids or the indicated IRF7-truncated mutantexpressing plasmids (IRF7DDBD, IRF7DAD, IRF7DID, and IRF7DRD) and M-HAexpressing plasmid or empty vector. The cells were collected and lysed at 36 hpt, and then immunoprecipitated with anti-HA Ab (IP: HA). The WCLs and IP complexes were analyzed by Western blotting with the indicated Abs. All experiments were repeated three times with similar results. Data were representative of the three independent experiments.

Article Snippet: For virus infection assays, Vero cells were infected with PEDV (multiplicity of infection [MOI] of 1) at 37 C for 18 h. The PEDV-infected or mock-infected cells were prepared as described above and the primary Abs were Rabbit antiPEDV M and mouse anti-IRF7 Ab (Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, Control, Transfection, Staining, Infection, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection.

doi: 10.4049/jimmunol.2001186

Figure Lengend Snippet: FIGURE 6. PEDV M protein inhibited the phosphorylation and dimerization of IRF7. (A) HEK-293T cells were transfected with pcDNA3.1-M-HA or empty vector plasmids, respectively. At 24 hpt, the cells were mock-infected or infected with SeV for another 16 h. The cells lysates were subjected to Western blotting analysis using the indicated Abs. (B and C) M protein inhibited IKKe/TBK1 induced IRF7 phosphorylation. HEK-293T cells were cotransfected with HA-IKKe (B) or HA-TBK1- (C) expressing plasmids and HA-IRF7 expressing plasmids, along with pcDNA3.1-M-HA or empty vector. The WCLs were collected for im- munoblotting analysis with the indicated Abs at 24 hpt. (D) HEK-293T cells were transfected with plasmids expressing M protein, M (F5S) protein, or empty vec- tor respectively. At 24 hpt, the cells were mock-infected or infected with SeV for 16 h. The cell lysates were subjected to Western blotting analysis using the indicated Abs. (E) PK-15 cells were transfected with poly (I:C), and the cells were infected with PEDV (MOI of 1) at 12 hpt. The cells were then incubated with the virus for 24 h or 36 h. The cell lysates were subjected to Western blotting with the indicated Abs. The change in abundance of pIRF7 was determined by den- sitometric analysis and normalized to IRF7 using the Image J software. (F) HEK-293T cells were transfected with pcDNA3.1-M-HA or empty vector plasmids re- spectively. At 24 hpt, the cells were mock-infected or infected with SeV for 16 h. The cells lysates were subjected to Western blotting analysis using the indicated Abs. (G) HEK-293T cells were cotransfected with HA-IRF7-, Flag-IRF7-expressing plasmids, and M-myc-expressing plasmids or empty vector. The cell lysates were immunoprecipitated with anti-Flag Ab (IP: Flag). The WCLs and IP complexes were analyzed by Western blotting using the indicated Abs. (H) HEK-293T cells were cotransfected with HA-TBK1-, Flag-IRF7-expressing plasmids, and M-myc-expressing plasmids or empty vector. The cell lysates were immunoprecipi- tated with anti-HA Ab (IP: HA). The WCLs and IP complexes were subjected to Western blotting analysis using the indicated Abs. (I) HEK-293T cells were co- transfected with HA-IKKe-, Flag-IRF7expressing plasmids, and M-myc-expressing plasmids or empty vector. The cell lysates were immunoprecipitated with anti-HA Ab (IP: HA). The WCLs and IP complexes were subjected to Western blotting analysis using the indicated Abs. All experiments were repeated three times with similar results. Data were representative of the three independent experiments.

Article Snippet: For virus infection assays, Vero cells were infected with PEDV (multiplicity of infection [MOI] of 1) at 37 C for 18 h. The PEDV-infected or mock-infected cells were prepared as described above and the primary Abs were Rabbit antiPEDV M and mouse anti-IRF7 Ab (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Infection, Western Blot, Expressing, Incubation, Virus, Software, Immunoprecipitation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection.

doi: 10.4049/jimmunol.2001186

Figure Lengend Snippet: FIGURE 8. PEDV M protein interacted with MyD88 and IRF7. (A) HEK-293T cells were cotransfected with M-HAexpressing plasmids and the indicat- ed Flag-tagged adaptor molecule (TLR7, IRAK1, IRAK4, IKKa, TLR3, or TRIF)expressing plasmids. The cells were collected at 36 hpt, and the lysates were immunoprecipitated by anti-Flag Ab (IP: Flag) or IgG control Ab (IP: Ig). The WCLs and IP complexes were analyzed by Western blotting with the in- dicated Abs. The experiments were repeated three times with similar results. Data were representative of the three independent experiments. (B) HEK-293T cells were cotransfected with M-mycexpressing plasmids and the indicated HA-tagged adaptor molecule (MyD88, TRAF6 or IRF7)expressing plasmids. The cells were collected at 36 hpt, and the lysates were immunoprecipitated by anti-HA Ab (IP: HA) or IgG control Ab (IP: Ig). The WCLs and IP com- plexes were analyzed by Western blotting with the indicated Abs. The experiments were repeated three times with similar results. Data were representative of the three independent experiments. (C) HEK-293T cells were cotransfected with M-HA and myc-TRAF3expressing plasmids. The cells were collected at 36 hpt, and the lysates were immunoprecipitated by anti-myc Ab (IP: myc) or IgG control Ab (IP: Ig). The WCLs and IP complexes were analyzed by West- ern blotting with the indicated Abs. The experiments were repeated three times with similar results. Data were representative of the three independent experi- ments. (D) HEK-293T cells were transfected with 1 mg of empty vector plasmids or TRAF3-expressing plasmids, together with 1.5 mg of plasmids expressing M protein or empty vector. At 24 hpt, the mRNA expression levels of IFN-a1, IFN-a2, and IFN-b were measured by qPCR. Results were repre- sentative of three independent experiments performed in triplicates. Graphs show mean ± SD. The statistical significance was calculated by the Student t test. **p < 0.01.

Article Snippet: For virus infection assays, Vero cells were infected with PEDV (multiplicity of infection [MOI] of 1) at 37 C for 18 h. The PEDV-infected or mock-infected cells were prepared as described above and the primary Abs were Rabbit antiPEDV M and mouse anti-IRF7 Ab (Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Control, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection.

doi: 10.4049/jimmunol.2001186

Figure Lengend Snippet: FIGURE 10. PEDV M protein promoted PEDV replication. (AC) PK-15 cells were transfected with pcDNA3.1-M-HA (1.5 mg or 3 mg) or empty vector. At 24 hpt, the cells were infected with PEDV at an MOI of 1 for 18 h. The cells were then collected and subjected to Western blotting (A) and qPCR analysis (B). The virus yields were determined by 50% tissue cultureinfective dose assay (C). Results were representative of three independent experiments performed in triplicates. Graphs show mean ± SD. The statistical significance was calculated by the Student t test. (D) Schematic diagram of the model that PEDV M protein targets IRF7 to inhibit innate immune response. PEDV M protein negatively regulates RIG-I, TLR3-, and TLR7-mediated type I IFN production by inhibiting the phosphorylation and dimerization of IRF7, leading to the decreased expression of type I IFN and various antiviral genes, which in turn promoted PEDV replication. PEDV also interacts with MyD88, whereas the biological effect remains unknown. *p < 0.05.

Article Snippet: For virus infection assays, Vero cells were infected with PEDV (multiplicity of infection [MOI] of 1) at 37 C for 18 h. The PEDV-infected or mock-infected cells were prepared as described above and the primary Abs were Rabbit antiPEDV M and mouse anti-IRF7 Ab (Santa Cruz Biotechnology).

Techniques: Transfection, Plasmid Preparation, Infection, Western Blot, Virus, Phospho-proteomics, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Voltage-Gated Proton Channel Hv1 Controls TLR9 Activation in Plasmacytoid Dendritic Cells.

doi: 10.4049/jimmunol.2000404

Figure Lengend Snippet: FIGURE 3. Hvcn12/2 mice show reduced pDC activation after challenge with CpGA. WT (n = 7) and Hvcn12/2 (n = 8) mice were injected i.v. with CpGA conjugated with DOTAP. Spleens were collected 8 h after injection. (A and B) Representative FACS plots and quantification of the percentages of CD317+ pDCs among CD45+ living cells (A) and of PDC-TREM expression among the CD317+ population (B). (C) Quantitative RT-PCR gene expression analysis of Irf7, Mx1, and Oas3 in WT (n = 5–6) and Hvcn12/2 (n = 4–6) pDCs sorted from the spleens of the same CpGA-treated mice. Data presented as mean 6 SEM and analyzed by multiple Student t test, using the Holm–Sidak method to determine statistical significance (*p # 0.05, **p # 0.01).

Article Snippet: Cells were stained with phospho-IRF7 Ab (1:300, D6M2I; Cell Signaling Technology) and species-specific Ab in AF555 (1:2000; Abcam).

Techniques: Activation Assay, Injection, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Melanoma suppression by quercein is correlated with RIG-I and type I interferon signaling.

doi: 10.1016/j.biopha.2020.109984

Figure Lengend Snippet: Fig. 3. Quercetin activated RIG-I signaling and induced IFN-I. (A) Confocal images were captured after quercetin treated for 24 h in B16 cells, stained with DAPI (blue) or RIG-I (green) (n = 3). (B) B16 cells were treated with quercetin or DMSO. Cell lysates were immunoblotted with anti-RIG-I, anti-STAT1 and anti-IRF7. GAPDH was used as a loading control. (C) Quantitative of RIG-I, STAT1 and IRF7. Showed as fold of GAPDH (n = 3). (D) qPCR analysis of DDX58, IFN-α and IFN-β in B16 cells treated with quercetin for 24 h (n = 3), normalized by GAPDH respectively. (E) Luciferase activity in lysates of 293 T cells transfected with luciferase report plasmids of pGL3-basic empty vector, RIG-I promoter, and luc-IFNβ, with quercetin treated or without (DMSO). Results are presented as the ratio of firefly luciferase to Renilla luciferase activity. Bar graphs represent the mean ± SEM (n = 5). (ns: no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Mouse anti-GAPDH antibody (Proteintech, China), rabbit anti-RIG-I Ab (Abcam, UK), rabbit anti-IRF7 Ab (Abcam, UK), rabbit anti-STAT1 Ab (CST, USA), rabbit anti-p-IRF7 Ab (CST, USA), rabbit anti-p-STAT1 Ab (CST, USA), rabbit anti-IFN-β Ab (Abcam, UK), rabbit anti-p65 Ab (CST, USA), rabbit antip-p65 Ab (CST, USA), rabbit anti-IKKε (CST, USA), HRP-labeled goat anti-mouse IgG(H + L) (Beyotime, China), and HRP-labeled goat antirabbit IgG(H + L) (Beyotime, China) were used.

Techniques: Staining, Control, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Melanoma suppression by quercein is correlated with RIG-I and type I interferon signaling.

doi: 10.1016/j.biopha.2020.109984

Figure Lengend Snippet: Fig. 4. Quercetin inhibited proliferation and promoted apoptosis in human A375 cell line. (A) Cell availability were analysis of by CCK8 after quercetin treated for 24 h and 48 h. Bar graphs represent the viability compared with control (n = 5). (B) Apoptosis analysis of A375 cells treated with quercetin for 24 h. Cells were collected and stained with PI and FITC-Annexin V, representative contour plots were shown (n = 3). (C) Bar graphs represent the apoptosis cells in each group. (D) qPCR analysis of DDX58, IFN-α and IFN-β in A375 cells treated with quercetin for 24 h (n = 3), normalized by GAPDH respectively. (E) Western-blot analysis expression of RIG-I, IRF7, p-IRF7, STAT1, and p-STAT1 in A375 cell lysates. (F) Quantitative of RIG-I, STAT1 and IRF7. Showed as fold of GAPDH (n = 3). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Mouse anti-GAPDH antibody (Proteintech, China), rabbit anti-RIG-I Ab (Abcam, UK), rabbit anti-IRF7 Ab (Abcam, UK), rabbit anti-STAT1 Ab (CST, USA), rabbit anti-p-IRF7 Ab (CST, USA), rabbit anti-p-STAT1 Ab (CST, USA), rabbit anti-IFN-β Ab (Abcam, UK), rabbit anti-p65 Ab (CST, USA), rabbit antip-p65 Ab (CST, USA), rabbit anti-IKKε (CST, USA), HRP-labeled goat anti-mouse IgG(H + L) (Beyotime, China), and HRP-labeled goat antirabbit IgG(H + L) (Beyotime, China) were used.

Techniques: Control, Staining, Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Melanoma suppression by quercein is correlated with RIG-I and type I interferon signaling.

doi: 10.1016/j.biopha.2020.109984

Figure Lengend Snippet: Fig. 6. Quercetin induced IFN-I by activating RIG-I and augmented by STAT1. (A) qPCR analysis of RIG-I signaling molecules and some ISGs in B16 cell lysates (blue), silencing with RIG-I siRNA (green), silencing with STAT1 (orange), and silencing with RIG-I and STAT1 (red) (n = 3). (B) Western-blot analysis expression of RIG-I, STAT1, and IRF7 in B16 cell lysates after silencing RIG-I. GAPDH was used as a loading control. (C) Quantitative of RIG-I, IRF7 and STAT1. Showed as fold of GAPDH (n = 3). (D) Apoptosis analysis of B16 cells after silencing RIG-I (n = 3). (E) Bar graphs represent the mean percentage of apoptosis cells.

Article Snippet: Mouse anti-GAPDH antibody (Proteintech, China), rabbit anti-RIG-I Ab (Abcam, UK), rabbit anti-IRF7 Ab (Abcam, UK), rabbit anti-STAT1 Ab (CST, USA), rabbit anti-p-IRF7 Ab (CST, USA), rabbit anti-p-STAT1 Ab (CST, USA), rabbit anti-IFN-β Ab (Abcam, UK), rabbit anti-p65 Ab (CST, USA), rabbit antip-p65 Ab (CST, USA), rabbit anti-IKKε (CST, USA), HRP-labeled goat anti-mouse IgG(H + L) (Beyotime, China), and HRP-labeled goat antirabbit IgG(H + L) (Beyotime, China) were used.

Techniques: Western Blot, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Melanoma suppression by quercein is correlated with RIG-I and type I interferon signaling.

doi: 10.1016/j.biopha.2020.109984

Figure Lengend Snippet: Fig. 7. Systemic diagram of the mechanism of quercetin. Quercetin acts as a RIG-I agonist. Activation of RIG-I leads to phosphorylation of IRF7. Phosphorylated dimers of IRF7 translocate to the nucleus, where they bind to and activate specific promoters, triggering expression of IFN-I as well as a subset of ISGs, which could furtherly activate the JAK1-STAT1 and amplify the RIG-I signaling.

Article Snippet: Mouse anti-GAPDH antibody (Proteintech, China), rabbit anti-RIG-I Ab (Abcam, UK), rabbit anti-IRF7 Ab (Abcam, UK), rabbit anti-STAT1 Ab (CST, USA), rabbit anti-p-IRF7 Ab (CST, USA), rabbit anti-p-STAT1 Ab (CST, USA), rabbit anti-IFN-β Ab (Abcam, UK), rabbit anti-p65 Ab (CST, USA), rabbit antip-p65 Ab (CST, USA), rabbit anti-IKKε (CST, USA), HRP-labeled goat anti-mouse IgG(H + L) (Beyotime, China), and HRP-labeled goat antirabbit IgG(H + L) (Beyotime, China) were used.

Techniques: Activation Assay, Phospho-proteomics, Expressing