anti phospho irf3  (Bioss)

Journal: Microbiology Spectrum

Article Title: Gallic acid inhibits EMCV infection via targeting the interaction between TBK1 and IRF3 to promote IFN-β expression

doi: 10.1128/spectrum.01524-25

Figure Lengend Snippet: GA regulates the RLR signaling pathway to inhibit EMCV replication. ( A ) Compared with the cells treated with 0 µM GA, those treated with GA (80/160 µM) significantly upregulated the expression of TBK1, p-TBK1, IRF3, and p-IRF3. ( B–D ) The overexpression of TBK1 and IRF3 significantly increased the expression of IFN-β. GAPDH was used as a control. Data were presented as the mean ± standard deviation (SD) of three independent experiments with P values indicating the results of one-way ANOVA and Tukey’s multiple comparison test or two-tailed t tests. *** P < 0.001, ** P < 0.01, and * P < 0.05 vs EMCV group; ### P < 0.001, ## P < 0.01, and # P < 0.05 vs mock group.

Article Snippet: TIFIH1/MDA5 polyclonal antibody (21775-1-AP), MAVS polyclonal antibody (14341-1-AP), and IRF3 polyclonal antibody (11312-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Over Expression, Control, Standard Deviation, Comparison, Two Tailed Test

Journal: Microbiology Spectrum

Article Title: Gallic acid inhibits EMCV infection via targeting the interaction between TBK1 and IRF3 to promote IFN-β expression

doi: 10.1128/spectrum.01524-25

Figure Lengend Snippet: GA induces the expression of IFN-β by targeting IRF3 to promote the interaction between TBK1 and IRF3. ( A ) GA (160 µM) significantly upregulated the expressions of TBK1, p-TBK1, IRF3, and p-IRF3. ( B ) GA (160 µM) significantly upregulated the expression of IFN-β. ( C ) GA could promote the interaction between TBK1 and IRF3. ( D ) siRNA targeting IRF3 was determined by Western blotting. After siRNA targeting IRF3, GA had no significant effect on the virus titer ( E ) and the expression of IFN-β ( F ) during EMCV infection. Data were presented as the mean ± standard deviation (SD) of three independent experiments, and P values indicate the results of one-way ANOVA with Tukey’s multiple comparison test or two-tailed t tests. *** P < 0.001, ** P < 0.01, and * P < 0.05 vs EMCV group; ### P < 0.001, ## P < 0.01, and # P < 0.05 vs mock group.

Article Snippet: TIFIH1/MDA5 polyclonal antibody (21775-1-AP), MAVS polyclonal antibody (14341-1-AP), and IRF3 polyclonal antibody (11312-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Western Blot, Virus, Infection, Standard Deviation, Comparison, Two Tailed Test

Journal: Microbiology Spectrum

Article Title: Gallic acid inhibits EMCV infection via targeting the interaction between TBK1 and IRF3 to promote IFN-β expression

doi: 10.1128/spectrum.01524-25

Figure Lengend Snippet: Schematic diagram showing that GA targets TBK1/IRF3 in vitro , promotes the expression of IFN-β, inhibits EMCV, and exhibits a protective effect on mice in vivo .

Article Snippet: TIFIH1/MDA5 polyclonal antibody (21775-1-AP), MAVS polyclonal antibody (14341-1-AP), and IRF3 polyclonal antibody (11312-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: In Vitro, Expressing, In Vivo

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

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