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IRF1 interacts with IRF3 and promotes IRF3 recruitment to ISG promoters . (A) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 5), and WCLs were analyzed by immunoblotting at 8 h post-infection. (B) THP-1 cells were mock-infected or infected with HSV-1 (MOI = 5), and nuclear and cytoplasmic fractions were isolated at the indicated time points and analyzed by immunoblotting. (C) HEK293T cells were transfected with the indicated plasmids, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose. The input and precipitated samples were analyzed by immunoblotting. (D) HT1080 cells were infected with HSV-1 (MOI = 10) for 8 h. Co-immunoprecipitation was performed with the indicated antibodies, followed by immunoblotting analysis. (E) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 10), and nuclear and cytoplasmic fractions were isolated at 8 h post-infection and analyzed by immunoblotting. (F) THP-1 cells were mock-infected or infected with HSV-1 (MOI = 10) for 5 or 10 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed with the indicated antibodies. (G) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 10) for 8 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed using an anti-IRF3 <t>polyclonal</t> antibody, and the input samples were also analyzed using an anti-IRF1 monoclonal antibody. (H) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were infected with HSV-1 (MOI = 10) for 10 h, followed by chromatin immunoprecipitation (ChIP) using an anti-IRF3 antibody or control IgG. IRF3 occupancy at the IFNB1 and IFNL1 promoter regions was assessed by qPCR.
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IRF1 interacts with IRF3 and promotes IRF3 recruitment to ISG promoters . (A) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 5), and WCLs were analyzed by immunoblotting at 8 h post-infection. (B) THP-1 cells were mock-infected or infected with HSV-1 (MOI = 5), and nuclear and cytoplasmic fractions were isolated at the indicated time points and analyzed by immunoblotting. (C) HEK293T cells were transfected with the indicated plasmids, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose. The input and precipitated samples were analyzed by immunoblotting. (D) HT1080 cells were infected with HSV-1 (MOI = 10) for 8 h. Co-immunoprecipitation was performed with the indicated antibodies, followed by immunoblotting analysis. (E) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 10), and nuclear and cytoplasmic fractions were isolated at 8 h post-infection and analyzed by immunoblotting. (F) THP-1 cells were mock-infected or infected with HSV-1 (MOI = 10) for 5 or 10 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed with the indicated antibodies. (G) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 10) for 8 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed using an anti-IRF3 polyclonal antibody, and the input samples were also analyzed using an anti-IRF1 monoclonal antibody. (H) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were infected with HSV-1 (MOI = 10) for 10 h, followed by chromatin immunoprecipitation (ChIP) using an anti-IRF3 antibody or control IgG. IRF3 occupancy at the IFNB1 and IFNL1 promoter regions was assessed by qPCR.

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: IRF1 interacts with IRF3 and promotes IRF3 recruitment to ISG promoters . (A) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 5), and WCLs were analyzed by immunoblotting at 8 h post-infection. (B) THP-1 cells were mock-infected or infected with HSV-1 (MOI = 5), and nuclear and cytoplasmic fractions were isolated at the indicated time points and analyzed by immunoblotting. (C) HEK293T cells were transfected with the indicated plasmids, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose. The input and precipitated samples were analyzed by immunoblotting. (D) HT1080 cells were infected with HSV-1 (MOI = 10) for 8 h. Co-immunoprecipitation was performed with the indicated antibodies, followed by immunoblotting analysis. (E) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 10), and nuclear and cytoplasmic fractions were isolated at 8 h post-infection and analyzed by immunoblotting. (F) THP-1 cells were mock-infected or infected with HSV-1 (MOI = 10) for 5 or 10 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed with the indicated antibodies. (G) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 10) for 8 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed using an anti-IRF3 polyclonal antibody, and the input samples were also analyzed using an anti-IRF1 monoclonal antibody. (H) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were infected with HSV-1 (MOI = 10) for 10 h, followed by chromatin immunoprecipitation (ChIP) using an anti-IRF3 antibody or control IgG. IRF3 occupancy at the IFNB1 and IFNL1 promoter regions was assessed by qPCR.

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Transduction, Control, Infection, Western Blot, Isolation, Transfection, Immunoprecipitation, Labeling, Chromatin Immunoprecipitation

IRF1 promotes antiviral innate immunity through its DNA-binding activity . (A) HEK293T cells were transfected with the indicated plasmids, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose. The input and immunoprecipitated samples were analyzed by immunoblotting. (B–E) THP-1 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with HSV-1 (MOI = 5). The indicated genes were quantified by RT-qPCR (B–D), and WCLs were analyzed by immunoblotting at 8 h post-infection (E). (F) THP-1 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with HSV-1 (MOI = 10) for 8 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed by immunoblotting using an anti-IRF3 polyclonal antibody, and the input samples were also analyzed using an anti-IRF1 monoclonal antibody. (G–K) HT1080 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with VSV (MOI = 5) for 8 h. The expression levels of the indicated genes were quantified by RT-qPCR.

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: IRF1 promotes antiviral innate immunity through its DNA-binding activity . (A) HEK293T cells were transfected with the indicated plasmids, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose. The input and immunoprecipitated samples were analyzed by immunoblotting. (B–E) THP-1 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with HSV-1 (MOI = 5). The indicated genes were quantified by RT-qPCR (B–D), and WCLs were analyzed by immunoblotting at 8 h post-infection (E). (F) THP-1 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with HSV-1 (MOI = 10) for 8 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed by immunoblotting using an anti-IRF3 polyclonal antibody, and the input samples were also analyzed using an anti-IRF1 monoclonal antibody. (G–K) HT1080 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with VSV (MOI = 5) for 8 h. The expression levels of the indicated genes were quantified by RT-qPCR.

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Binding Assay, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Control, Infection, Quantitative RT-PCR, Labeling