Journal: Frontiers in Immunology

Article Title: Mfn2 is responsible for inhibition of the RIG-I/IRF7 pathway and activation of NLRP3 inflammasome in Seneca Valley virus-infected PK-15 cells to promote viral replication

doi: 10.3389/fimmu.2022.955671

Figure Lengend Snippet: IRF7 inhibitor (BX795) inhibited IRF7 phosphorytion and IFN-λ3 secretion thus promote SVV proliferation. (A) The western blot assay for p-IRF7. (B) The relative protein expression level of IRF3. (C, D) The Elisa assay for IFN-λ1 and IFN-λ3. (E) The qRT-PCR assay for SVV copy number. One-way analysis of variance (ANOVA) were used to investigate the significance of differences between the SVV groups and the SVV+BX795 group or between the control group and SVV group *; P < 0.05, **; P < 0.01, ***; P < 0.005, ****; P < 0.001, ns means no significance.

Article Snippet: The primary antibodies were RIG-I polyclonal antibody (CST, United States), anti-Phospho-IRF7 (Bioss, China), GAPDH (Abcam, United States), Rabbit Anti-ASC antibody (Proteintech, United States), cleaved Rabbit Anti-IL-1β antibody (CST, United States), Rabbit Anti-IL-18 antibody (CST, United States), Rabbit Caspase 1/p20/p10 Polyclonal antibody (proteintech, United States); Mfn2 polyclonal antibody (proteintech, United States) and NLRP3 polyclonal antibody (proteintech, United States).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Mfn2 is responsible for inhibition of the RIG-I/IRF7 pathway and activation of NLRP3 inflammasome in Seneca Valley virus-infected PK-15 cells to promote viral replication

doi: 10.3389/fimmu.2022.955671

Figure Lengend Snippet: Mfn2 overexpression restrain SVV-induced RIG-I/IRF7 protein expression level and IFN-λ3 content in supernatant in PK-15 cells (A) The western blot assay for RIG-I and p-IRF7. (B, C) The relative protein expression level of RIG-I and p-IRF7. (D) The Elisa assay for IFN-λ3 detection. Unpaired T-test and One-way analysis of variance (ANOVA) were used to investigate the significance of differences. * means the difference between the WT and Ad-Mfn2 in control group; # means the difference between the WT and Ad-Mfn2 in SVV group. *; P < 0.05, ** ,## ; P < 0.01, *** ,### ; P < 0.005, #### ; P < 0.001, ns means no significance.

Article Snippet: The primary antibodies were RIG-I polyclonal antibody (CST, United States), anti-Phospho-IRF7 (Bioss, China), GAPDH (Abcam, United States), Rabbit Anti-ASC antibody (Proteintech, United States), cleaved Rabbit Anti-IL-1β antibody (CST, United States), Rabbit Anti-IL-18 antibody (CST, United States), Rabbit Caspase 1/p20/p10 Polyclonal antibody (proteintech, United States); Mfn2 polyclonal antibody (proteintech, United States) and NLRP3 polyclonal antibody (proteintech, United States).

Techniques: Over Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Mfn2 is responsible for inhibition of the RIG-I/IRF7 pathway and activation of NLRP3 inflammasome in Seneca Valley virus-infected PK-15 cells to promote viral replication

doi: 10.3389/fimmu.2022.955671

Figure Lengend Snippet: Schematic diagram of the possible mechanism of Mfn2 antiviral response. Mfn2 promote SVV proliferation mostly via down-regulating RIG-I/IRF7-dependent IFN-λ3 protein expression levels.

Article Snippet: The primary antibodies were RIG-I polyclonal antibody (CST, United States), anti-Phospho-IRF7 (Bioss, China), GAPDH (Abcam, United States), Rabbit Anti-ASC antibody (Proteintech, United States), cleaved Rabbit Anti-IL-1β antibody (CST, United States), Rabbit Anti-IL-18 antibody (CST, United States), Rabbit Caspase 1/p20/p10 Polyclonal antibody (proteintech, United States); Mfn2 polyclonal antibody (proteintech, United States) and NLRP3 polyclonal antibody (proteintech, United States).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Chronic Oral Infection with Porphyromonas gingivalis Accelerates Atheroma Formation by Shifting the Lipid Profile

doi: 10.1371/journal.pone.0020240

Figure Lengend Snippet: Cells were stimulated with 1.0 µg/ml of P. gingivalis LPS, 0.1 µg/ml of E. coli LPS or 0.1 µg/ml of Pam3CSK4 for 12 hrs. Phosphorylation of IRF3 cellular extracts was analyzed by western blotting. Blot is a representative of three independent experiments.

Article Snippet: Rabbit anti-mouse IRF3 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse phospho-IRF3 (Cell Signaling Technology, Danvers, MA), rabbit anti-mouse GAPDH (Santa Cruz Biotechnology), peroxidase labeled anti-rabbit antibody (GE Healthcare, Munich, Germany) and ECL Plus Western Blotting Reagent Pack (Amersham Biosciences, Buckinghamshire, UK) were used for western blotting experiments.

Techniques: Phospho-proteomics, Western Blot

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: iScience

Article Title: Mycobacterial CpsA activates type I IFN signaling in macrophages via cGAS-mediated pathway

doi: 10.1016/j.isci.2024.109807

Figure Lengend Snippet: CpsA-related type I IFN production is through cGAS-TBK1-IRF3 pathway (A) BMDMs extracted from WT mice or Cgas knockout mice were infected with WT or Δ cpsA Mm strains at MOI of 10. RNA was isolated at 10 h after infection. Ifnβ levels were determined by RT-qPCR in different infection cells; values were normalized to Gapdh and uninfected BMDMs. (B) RAW264.7 cells were infected with WT and Δ cpsA strains at MOI of 10. Total cell lysates and nuclear part were harvested at indicated time points. Phospho-TBK1 and TBK1 protein levels were determined by using a densitometer and (C) corresponding statistical results were calculated from two independent experiments by ImageJ software, GAPDH as a reference protein. (D) The protein levels of IRF3 from nuclear fraction were determined by western blot (WB) and (E) corresponding statistical results were calculated from two independent experiments by ImageJ software, H3 as a reference. (F) Nuclear p65 and H3 protein levels were determined by WB. Shown is a representative experiment of three. Data represent means ± SD of three independent experiments. A, ∗ p < 0.05, ns, not significant by unpaired t test, and C, D ∗ p < 0.05, ∗∗ p < 0.01, ns, not significant by two-way ANOVA with multiple comparisons test.

Article Snippet: IRF3 Rabbit pAb , ABclonal , Cat#A2172; RRID:AB_2764190.

Techniques: Knock-Out, Infection, Isolation, Quantitative RT-PCR, Software, Western Blot

Journal: iScience

Article Title: Mycobacterial CpsA activates type I IFN signaling in macrophages via cGAS-mediated pathway

doi: 10.1016/j.isci.2024.109807

Figure Lengend Snippet:

Article Snippet: IRF3 Rabbit pAb , ABclonal , Cat#A2172; RRID:AB_2764190.

Techniques: Produced, Virus, Fluorescence, Recombinant, Modification, Lysis, Western Blot, Protein Extraction, LDH Cytotoxicity Assay, Purification, Bicinchoninic Acid Protein Assay, 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 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: The commercial Abs used in this study included an anti-Flag mouse mAb (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag rabbit mAb (Invitrogen), anti-myc mouse mAb (Santa Cruz Biotechnology), anti-HA mouse mAb (Invitrogen), anti-HA rabbit mAb (Invitrogen), anti-IRF7 mouse mAb (Santa Cruz Biotechnology), anti-IRF7 rabbit mAb (Boster), anti-IRF7 rabbit polyclonal Ab (BioWorld), anti-pIRF7 rabbit mAb (Cell Signaling Technology), anti-IRF3 rabbit mAb (Cell Signaling Technology), anti-pIRF3 rabbit mAb (Cell Signaling Technology), anti b-actin mouse polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX), anti PEDV-M rabbit polyclonal Ab, which was kindly provided by Dr. Guangliang Liu (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), and anti PEDV-N mouse mAb (YouLong Biotech).

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: The commercial Abs used in this study included an anti-Flag mouse mAb (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag rabbit mAb (Invitrogen), anti-myc mouse mAb (Santa Cruz Biotechnology), anti-HA mouse mAb (Invitrogen), anti-HA rabbit mAb (Invitrogen), anti-IRF7 mouse mAb (Santa Cruz Biotechnology), anti-IRF7 rabbit mAb (Boster), anti-IRF7 rabbit polyclonal Ab (BioWorld), anti-pIRF7 rabbit mAb (Cell Signaling Technology), anti-IRF3 rabbit mAb (Cell Signaling Technology), anti-pIRF3 rabbit mAb (Cell Signaling Technology), anti b-actin mouse polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX), anti PEDV-M rabbit polyclonal Ab, which was kindly provided by Dr. Guangliang Liu (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), and anti PEDV-N mouse mAb (YouLong Biotech).

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: The commercial Abs used in this study included an anti-Flag mouse mAb (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag rabbit mAb (Invitrogen), anti-myc mouse mAb (Santa Cruz Biotechnology), anti-HA mouse mAb (Invitrogen), anti-HA rabbit mAb (Invitrogen), anti-IRF7 mouse mAb (Santa Cruz Biotechnology), anti-IRF7 rabbit mAb (Boster), anti-IRF7 rabbit polyclonal Ab (BioWorld), anti-pIRF7 rabbit mAb (Cell Signaling Technology), anti-IRF3 rabbit mAb (Cell Signaling Technology), anti-pIRF3 rabbit mAb (Cell Signaling Technology), anti b-actin mouse polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX), anti PEDV-M rabbit polyclonal Ab, which was kindly provided by Dr. Guangliang Liu (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), and anti PEDV-N mouse mAb (YouLong Biotech).

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: The commercial Abs used in this study included an anti-Flag mouse mAb (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag rabbit mAb (Invitrogen), anti-myc mouse mAb (Santa Cruz Biotechnology), anti-HA mouse mAb (Invitrogen), anti-HA rabbit mAb (Invitrogen), anti-IRF7 mouse mAb (Santa Cruz Biotechnology), anti-IRF7 rabbit mAb (Boster), anti-IRF7 rabbit polyclonal Ab (BioWorld), anti-pIRF7 rabbit mAb (Cell Signaling Technology), anti-IRF3 rabbit mAb (Cell Signaling Technology), anti-pIRF3 rabbit mAb (Cell Signaling Technology), anti b-actin mouse polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX), anti PEDV-M rabbit polyclonal Ab, which was kindly provided by Dr. Guangliang Liu (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), and anti PEDV-N mouse mAb (YouLong Biotech).

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: The commercial Abs used in this study included an anti-Flag mouse mAb (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag rabbit mAb (Invitrogen), anti-myc mouse mAb (Santa Cruz Biotechnology), anti-HA mouse mAb (Invitrogen), anti-HA rabbit mAb (Invitrogen), anti-IRF7 mouse mAb (Santa Cruz Biotechnology), anti-IRF7 rabbit mAb (Boster), anti-IRF7 rabbit polyclonal Ab (BioWorld), anti-pIRF7 rabbit mAb (Cell Signaling Technology), anti-IRF3 rabbit mAb (Cell Signaling Technology), anti-pIRF3 rabbit mAb (Cell Signaling Technology), anti b-actin mouse polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX), anti PEDV-M rabbit polyclonal Ab, which was kindly provided by Dr. Guangliang Liu (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), and anti PEDV-N mouse mAb (YouLong Biotech).

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: The commercial Abs used in this study included an anti-Flag mouse mAb (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag rabbit mAb (Invitrogen), anti-myc mouse mAb (Santa Cruz Biotechnology), anti-HA mouse mAb (Invitrogen), anti-HA rabbit mAb (Invitrogen), anti-IRF7 mouse mAb (Santa Cruz Biotechnology), anti-IRF7 rabbit mAb (Boster), anti-IRF7 rabbit polyclonal Ab (BioWorld), anti-pIRF7 rabbit mAb (Cell Signaling Technology), anti-IRF3 rabbit mAb (Cell Signaling Technology), anti-pIRF3 rabbit mAb (Cell Signaling Technology), anti b-actin mouse polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX), anti PEDV-M rabbit polyclonal Ab, which was kindly provided by Dr. Guangliang Liu (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), and anti PEDV-N mouse mAb (YouLong Biotech).

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: The commercial Abs used in this study included an anti-Flag mouse mAb (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag rabbit mAb (Invitrogen), anti-myc mouse mAb (Santa Cruz Biotechnology), anti-HA mouse mAb (Invitrogen), anti-HA rabbit mAb (Invitrogen), anti-IRF7 mouse mAb (Santa Cruz Biotechnology), anti-IRF7 rabbit mAb (Boster), anti-IRF7 rabbit polyclonal Ab (BioWorld), anti-pIRF7 rabbit mAb (Cell Signaling Technology), anti-IRF3 rabbit mAb (Cell Signaling Technology), anti-pIRF3 rabbit mAb (Cell Signaling Technology), anti b-actin mouse polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX), anti PEDV-M rabbit polyclonal Ab, which was kindly provided by Dr. Guangliang Liu (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), and anti PEDV-N mouse mAb (YouLong Biotech).

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