Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

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

Article Snippet: Glutathione S-transferase ( GST )-Pulldown Assay Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Sequencing

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

Figure Lengend Snippet: ARV p17 interacts with IFI16. (A) The transformed competent cells grown on the SD/-His/-Leu/-Trp plate were used to demonstrate the negative and positive controls. (B) Coimmunoprecipitation assay was performed to analyze the interaction between IFI16 and ARV p17 protein. Vero cells were transfected with IFI16-Flag plasmid, infection with ARV for 48 h, and then harvested. Cell lysates were immunoprecipitated using antibody against Flag, followed by western blotting. (C) An in vitro GST pulldown assay was performed to determine whether p17 direct interacts with IFI16. All indicated proteins were examined by Western blotting with the respective antibodies. (D) Confocal microscopy analysis revealed the colocalization of IFI16 and ARV p17 protein in Vero cells cotransfected with IFI16-RED and p17-GFP for 24 h.

Article Snippet: Glutathione S-transferase ( GST )-Pulldown Assay Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Transformation Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Infection, Immunoprecipitation, Western Blot, In Vitro, GST Pulldown Assay, Confocal Microscopy

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

Figure Lengend Snippet: Identification of the interaction regions of p17 and IFI16 was performed in this study. (A and B) Schematic representation of the truncation mutants of p17 and IFI16 utilized in this investigation is shown, which mainly based on the amino acid assayed by domain analysis in the website: http://tools.immuneepitope.org/bcell/. (C) HEK-293T cells were co-transfected with HINa-Flag, HINb-Flag and p1719-146, p1761-146, p17119-146 constructs. After 36 h, cell lysates were subjected to immunoprecipitation followed by western blotting analysis. (D) Colocalization analysis of IFI16 with different domains of the p17 protein including p1719-146, p1761-146, and p17119-146 was conducted using confocal microscopy after cotransfection of HEK-293T cells with IFI16-red and p17-GFP for 24 h.

Article Snippet: Glutathione S-transferase ( GST )-Pulldown Assay Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Confocal Microscopy, Cotransfection

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

Figure Lengend Snippet: The expression of IFI16 is upregulated by p17 or ARV infection. (A) The transcript levels of ARV were examined at 0, 6, 12, 24, 36, and 48 hpi by qRT-PCR. (B) The protein levels of p17 expression were assessed during different time points of ARV infection. (C) Vero cells were infected with ARV at specific time intervals, and the mRNA levels of IFI16 were measured using qRT-PCR. (D) The protein levels of p17 expression were evaluated after transfection with the p17-pCDNA3.1 plasmid at various time points. (E) Quantification of IFI16 mRNA expression in Vero cells transfected with p17-pcDNA3.1 plasmids for different durations was performed using qRT-PCR. All experiments were repeated at least 3 times with similar results. * P < 0.05, ** P < 0.01.

Article Snippet: Glutathione S-transferase ( GST )-Pulldown Assay Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Expressing, Infection, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

Figure Lengend Snippet: The IFI16 protein exerts suppressive effects on ARV proliferation, as demonstrated by the following experiments. (A) Overexpression of IFI16 in Vero cells via transfection with plasmid IFI16-pcDNA3.1 was confirmed by qRT-PCR analysis of IFI16 expression levels. (B) The transcript quantity of ARV was assessed by qRT-PCR. (C) The protein level of p17 expression in indicated treatment group. (D) Vero cells were transfected with siIFI16 or a nontargeting control (siNC) for 24 h, the knockdown efficiency of IFI16 was examined by qRT-PCR. (E) The transcript quantity of ARV was assessed by qRT-PCR. (F) The protein level of p17 expression in indicated treatment group. All experiments were repeated at least 3 times with similar results. * P < 0.05, ** P < 0.01.

Article Snippet: Glutathione S-transferase ( GST )-Pulldown Assay Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

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

Article Snippet: Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Sequencing

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

Figure Lengend Snippet: ARV p17 interacts with IFI16. (A) The transformed competent cells grown on the SD/-His/-Leu/-Trp plate were used to demonstrate the negative and positive controls. (B) Coimmunoprecipitation assay was performed to analyze the interaction between IFI16 and ARV p17 protein. Vero cells were transfected with IFI16-Flag plasmid, infection with ARV for 48 h, and then harvested. Cell lysates were immunoprecipitated using antibody against Flag, followed by western blotting. (C) An in vitro GST pulldown assay was performed to determine whether p17 direct interacts with IFI16. All indicated proteins were examined by Western blotting with the respective antibodies. (D) Confocal microscopy analysis revealed the colocalization of IFI16 and ARV p17 protein in Vero cells cotransfected with IFI16-RED and p17-GFP for 24 h.

Article Snippet: Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Transformation Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Infection, Immunoprecipitation, Western Blot, In Vitro, GST Pulldown Assay, Confocal Microscopy

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

Figure Lengend Snippet: Identification of the interaction regions of p17 and IFI16 was performed in this study. (A and B) Schematic representation of the truncation mutants of p17 and IFI16 utilized in this investigation is shown, which mainly based on the amino acid assayed by domain analysis in the website: http://tools.immuneepitope.org/bcell/. (C) HEK-293T cells were co-transfected with HINa-Flag, HINb-Flag and p1719-146, p1761-146, p17119-146 constructs. After 36 h, cell lysates were subjected to immunoprecipitation followed by western blotting analysis. (D) Colocalization analysis of IFI16 with different domains of the p17 protein including p1719-146, p1761-146, and p17119-146 was conducted using confocal microscopy after cotransfection of HEK-293T cells with IFI16-red and p17-GFP for 24 h.

Article Snippet: Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Confocal Microscopy, Cotransfection

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

Figure Lengend Snippet: The expression of IFI16 is upregulated by p17 or ARV infection. (A) The transcript levels of ARV were examined at 0, 6, 12, 24, 36, and 48 hpi by qRT-PCR. (B) The protein levels of p17 expression were assessed during different time points of ARV infection. (C) Vero cells were infected with ARV at specific time intervals, and the mRNA levels of IFI16 were measured using qRT-PCR. (D) The protein levels of p17 expression were evaluated after transfection with the p17-pCDNA3.1 plasmid at various time points. (E) Quantification of IFI16 mRNA expression in Vero cells transfected with p17-pcDNA3.1 plasmids for different durations was performed using qRT-PCR. All experiments were repeated at least 3 times with similar results. * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Expressing, Infection, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: Poultry Science

Article Title: IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication

doi: 10.1016/j.psj.2024.103506

Figure Lengend Snippet: The IFI16 protein exerts suppressive effects on ARV proliferation, as demonstrated by the following experiments. (A) Overexpression of IFI16 in Vero cells via transfection with plasmid IFI16-pcDNA3.1 was confirmed by qRT-PCR analysis of IFI16 expression levels. (B) The transcript quantity of ARV was assessed by qRT-PCR. (C) The protein level of p17 expression in indicated treatment group. (D) Vero cells were transfected with siIFI16 or a nontargeting control (siNC) for 24 h, the knockdown efficiency of IFI16 was examined by qRT-PCR. (E) The transcript quantity of ARV was assessed by qRT-PCR. (F) The protein level of p17 expression in indicated treatment group. All experiments were repeated at least 3 times with similar results. * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant full-length glutathione S-transferase (GST)-p17 fusion protein and GST protein were expressed induction in Escherichia coli BL21 ( DE3 ) Strain (Invitrogen, Carlsbad, CA), while IFI16-Flag plasmid was transfected into Vero cells.

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing