Journal: Journal of Nanobiotechnology

Article Title: Apoptotic tumor cell-derived microparticles loading Napabucasin inhibit CSCs and synergistic immune therapy

doi: 10.1186/s12951-023-01792-8

Figure Lengend Snippet: In vivo validation of the mechanism of NAP inhibiting colon cancer. A The KEGG enrichment analysis indicated that the immune-related pathway was regulated after TMPs treatment. B GSEA algorithm was used to analyze the changes in the cytosolic DNA-sensing pathway after TMPs treatment. C p-TBK1(Ser172) and p-IRF3(Ser386), markers of activation of innate immune pathway cytosolic DNA-sensing, were detected after TMPs treatment. D – F RT-qPCR detected downstream target genes of the DNA-sensing pathway. G – H Representative flow analysis of CD3 + CD4 + T cells and CD3 + CD8 + T cells in tumor tissues. I – J Representative fluorescent images of CD3 + CD4 + T cells and CD3 + CD8 + T cells in tumor tissues. (n = 6, scale bars:100 μm)

Article Snippet: GAPDH antibody (#ab8245, 1:3000) was purchased from Abcam. p-TBK1(Ser172) antibody (#5483,1:1000), p-IRF3(Ser386) (#37829, 1:1000) were purchased from Cell Signaling Technology.

Techniques: In Vivo, Activation Assay, Quantitative RT-PCR

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 deficiency inhibits VSV-induced IKKε and IRF3 phosphorylation. a , b Immunoblot analysis of phosphorylated and total proteins in lysates of PMs obtained from WT (TAOK1 f/f ) and TAOK1cko (Lyz2 + TAOK1 f/f ) mice infected with VSV for indicated time. c Immunoblot analysis of phosphorylated and total proteins in lysates of Myc-TAOK1 overexpressing RAW264.7 macrophages infected with VSV for indicated time. d Luciferase activity in HEK293T cells transfected with Ifnb luciferase reporter, a Renilla-TK reporter, and an expression vector for TAOK1, along with plasmids encoding RIG-I N, TBK1, IKKε, and IRF3 5D. Data are representative of three independent experiments. Data are mean ± SD. * p < 0.05, ** p < 0.01 (Student's t test). ns, not significant.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

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

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 interacts with TBK1. a Mouse PMs (from wild-type [WT] C57BL/6 mice, 6–8 weeks old) were infected with VSV for indicated hours. Immunoblot analysis of endogenous signal adapters immunoprecipitated with antibody to TAOK1. b Immunoblot analysis of HEK293T cells that co-transfected with Myc-TAOK1 plus HA-TBK1 or HA-IRF3 plasmids, then immunoprecipitated with antibody to HA tag. c Immunoblot analysis of HEK293T cells that co-transfected with Myc-TAOK1 plus Flag-RIG-I plasmids, then immunoprecipitated with the antibody to Flag tag. d Immunoblot analysis of HEK293T cells that co-transfected with Myc-TAOK1 plus HA-MAVS plasmids, then immunoprecipitated with the antibody to HA tag. e WT and MAVS-deficient HEK293T (MAVS-KO) cells were infected with VSV for indicated time, followed by immunoprecipitation (IP) with anti-TAOK1 and immunoblot analysis with indicated antibodies. f Q-PCR analysis of IFNB1 and TNFA mRNA expression in MAVS-deficient HEK293T (MAVS-KO) cells transfected with TAOK1 expressing plasmid and infected with VSV for indicated time. Data are from one experiment of three similar results. Data are mean ± SD. * p < 0.05, ** p < 0.01 (Student's t test). WCL, whole-cell lysates; nd, not detected.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection, Western Blot, Immunoprecipitation, Transfection, FLAG-tag, Expressing, Plasmid Preparation

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 promotes TBK1-IRF3 complex formation. a HEK293T cells were transfected with plasmids encoding HA-TBK1, Flag-IRF3, and varying doses of a plasmid encoding Myc-TAOK1 (0.5, 1.0, and 1.5 μg). Cells were harvested 24 h after transfection for immunoblot analysis as indicated with the antibody to Flag tag. b HEK293T cells were transfected with plasmids encoding Flag-IKKε, HA-IRF3, and varying doses of a plasmid encoding Myc-TAOK1 (0.5, 1.0, and 1.5 μg). Cells were harvested 24 h after transfection for immunoblot analysis as indicated with antibody to HA tag. c Mouse PMs from WT (TAOK1 f/f ) and TAOK1cko (Lyz2 + TAOK1 f/f ) mice were infected with VSV for indicated hours. Immunoblot analysis of endogenous IRF3 immunoprecipitated with antibody to TBK1. Data are from one experiment of three similar results. * p < 0.05, ** p < 0.01. WCL, whole-cell lysates; ns, not significant.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, FLAG-tag, Infection, Immunoprecipitation

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 promotes the interaction between TBK1 and IRF3 by trafficking TBK1 along microtubules. a HeLa cells co-transfected with Flag-IRF3 and Myc-TAOK1 plasmids, then pretreated with DMSO or colchicine (10 μm) for 1 h, followed by infection with or without VSV for 4 h. Confocal microscopy of co-localization between Flag-IRF3 (green) and endogeneous TBK1 (red). DAPI served as a marker of nuclei (blue). Scale bar, 10 μm. b Confocal microscopy of HeLa cells transfected with Myc-TAOK1, Myc-TAOK1 K57A, Myc-TAOK1C, and Myc-TAOK1N plasmids (green) followed by VSV infection for 4 h α-tubulin (red) was used to probe the microtubules. DAPI served as a marker of nuclei (blue). Scale bar, 10 μm. c Immunoblot analysis of phosphorylated and total MAP4 proteins in lysates of PMs obtained from WT (TAOK1 f/f ) and TAOK1cko (Lyz2 + TAOK1 f/f ) mice infected with VSV for indicated time. d Mouse PMs were pretreated with DMSO or colchicine (10 μm) for 1 h and then infected with VSV for 4 h. Immunoblot analysis of phosphorylated and total IRF3 and MAP4 proteins in cell lysates. e Mouse PMs were pretreated with DMSO or colchicine (10 μM) for 1 h and then infected with VSV for 4 h. Immunoblot analysis of endogenous signal adapters immunoprecipitated with antibody to TAOK1. * p < 0.05, ** p < 0.01. Data are representative of three independent experiments with similar results. ns, not significant; WCL, whole-cell lysates.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Infection, Confocal Microscopy, Marker, Western Blot, Immunoprecipitation

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: A schematic diagram revealing the proposed mechanism by which TAOK1 positively regulates antiviral immune responses. (1) TAOK1 promoted virus-induced MAP4 phosphorylation and microtubule detachment; (2) TAOK1 bound more dynein instead of MAP4 upon virus infection and promoted the perinuclear transport of TBK1 along microtubules; (3) TAOK1 positively regulated antiviral signaling by promoting the TBK1-IRF3 interaction and IRF3 phosphorylation.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection

Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: (A) HEK-293T cells were transfected with 1 μg of empty vector or the indicated FLAG-2C-expressing plasmids, along with 1 μg of empty vector or HA-cGAS plus HA-STING expressing plasmids. At 24 hpt, the IFN-β protein amount in the supernatant was determined by ELISA, and the expression of 2C was confirmed by Western blotting. (B) STING-HEK-293T cells were transfected with increasing amount (0, 3, or 6 μg) of the indicated FLAG-2C-expressing plasmids. At 24 hpt, cells were transfected with poly(dA:dT) (2 μg/ml) for 12 h. The cells lysates were immunoprecipitated with anti-STING antibody. The antibody-antigen complexes were detected using anti-STING, TBK1, and FLAG antibodies, respectively. (C) Schematic representation of the structure and conserved functional sites in EV-A71 2C protein. The redder the color was, the more conservative the sites were. (D, E) HEK-293T cells were transfected with 1 μg of empty vector or EV-A71 (D), CA16 (E), and EMCV (E) FLAG-2C- or FLAG-2C mutants-expressing plasmids, along with 1 μg of empty vector or HA-cGAS plus HA-STING expressing plasmids. At 24 hpt, the IFN-β protein amount in the supernatant was determined by ELISA, and the expression of 2C was confirmed by Western blotting. (F) STING-HEK-293T cells were transfected with 6 μg of empty vector, FLAG-2C- or FLAG-2C mutants-expressing plasmids. At 24 hpt, cells were transfected with poly(dA:dT) (2 μg/ml) for 12 h. The cells lysates were immunoprecipitated with anti-STING antibody. The antibody-antigen complexes were detected using anti-STING, TBK1, and FLAG antibodies, respectively. (G) HEK-293T cells were transfected with 1 μg of empty vector, FLAG-2C- or FLAG-2C mutants-expressing plasmids, and 1 μg of HA-cGAS plus HA-STING expressing plasmids. At 24 hpt, expression of IRF3, p-IRF3, and FLAG-2C protein was determined by Western blotting. The IRF3 dimerization was detected using native PAGE. Error bars show standard deviation. **, P <0.01.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation, Functional Assay, Clear Native PAGE, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation

doi: 10.1128/spectrum.03413-22

Figure Lengend Snippet: aMPV P protein targets IRF7 for inhibiting IFN-β activation in DF-1 cells. (A and B) DF-1 cells were cotransfected with IRF7-luc or NF-κB-luc and pRL-TK, expression plasmid P (HA-P), or empty plasmid (HA). At 24 hpt, the cells were transfected with poly (I·C) for 12 h and subsequently subjected to luciferase activity measurement. The expressions of HA-tag protein and β-actin were detected by Western blotting. (C to F) DF-1 cells were cotransfected with MDA5, MAVS, TBK1, or IRF7 expression plasmids or empty plasmid (HA), IFN-β-luc, and pRL-TK. At 36 hpt, the cells were processed and the luciferase activity measured. The expression of HA-tag protein and Flag-tag protein were detected by Western blotting. Values are presented as the mean ± SD from three independent experiments. Statistical analysis was performed using Student's t test (ns, P > 0.05; ****, P < 0.0001).

Article Snippet: The commercial antibodies used in this study were as follows: rabbit anti-Flag antibody and rabbit anti-GFP antibody (HuaAn, China), rabbit anti-IRF3 antibody and rabbit anti-histone H3 antibody (ABclonal, China), rabbit anti-p-IRF3 antibody (CST, USA), rabbit anti-β-tubulin antibody (MBL, Japan), rabbit anti-HA antibody, mouse anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and HRP-conjugated anti-mouse secondary antibody (Sangon Biotech, China), and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, USA).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Western Blot, FLAG-tag

Journal: Microbiology Spectrum

Article Title: Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation

doi: 10.1128/spectrum.03413-22

Figure Lengend Snippet: aMPV P protein interacted with IRF7. (A and B) HEK-293T cells were cotransfected with GFP-P and Flag-IRF7 expression plasmids for 36 h. The cells were processed, immunoprecipitated with anti-GFP, anti-Flag, or anti-β-actin antibody, and subsequently analyzed using Western blotting with anti-Flag, anti-GFP, or anti-β-actin antibody. (C) DF-1 cells were cotransfected with GFP-P and/or Flag-IRF7 expression plasmids for 36 h, fixed, and processed. Dual labeling for GFP-P (green) and Flag-IRF7 (red) were observed by immunostaining with anti-Flag antibody. Cell nuclei were stained with DAPI (blue). The yellow observed in the merged images was considered to indicate colocalization areas. Scale bars, 20 μm. (D) DF-1 cells were transfected with Flag-IRF7 or Flag expression plasmids for 12 h, followed by infection with aMPV/C for 72 h. The cells were processed, immunoprecipitated with anti-Flag antibody, and subsequently analyzed using Western blotting with anti-Flag or anti-P antibody. (E) DF-1 cells were treated with Flag-IRF7 or Flag plasmids and aMPV/C as described in Fig. 3D , fixed, and processed. Dual labeling for P (red) and Flag- (green) were observed by immunostaining with anti-P or anti-Flag antibody. Cell nuclei were stained with DAPI (blue). The yellow observed in the merged images indicates colocalization areas. Scale bars, 20 μm. (F) Schematic representation of chicken IRF7 at different lengths, including full-length IRF7 (aa 1 to 492), IRF7 DBD (aa 1 to 143), IRF7 ΔDBD (aa 143 to 492), IRF7 ΔIRD (aa 1 to 303), IRF7 IRD (aa 303 to 492), and IRF7 AD (CAD+VAD) (aa 143 to 303). (G) HEK-293T cells were cotransfected with GFP-P and Flag-IRF7, IRF7 ΔDBD, or IRF7 ΔIRD expression plasmids for 36 h. The cells were processed, immunoprecipitated with anti-GFP or anti-Flag antibody, and analyzed using Western blotting with anti-Flag, anti-GFP, or anti-β-actin antibody. (H) HEK-293T cells were cotransfected with GFP-P and Flag-IRF7, IRF7 ΔIRD, IRF7 DBD, or IRF7 AD expression plasmids for 36 h. The cells were processed, immunoprecipitated with anti-GFP, anti-Flag, or anti-β-actin antibody, and analyzed using Western blotting with anti-Flag or anti-GFP antibody. (I) HEK-293T cells were cotransfected with GFP-P and Flag-IRF7, IRF7 ΔDBD, IRF7 AD, or IRF7 IRD expression plasmids for 36 h. The cells were processed, immunoprecipitated with anti-GFP or anti-Flag antibody, and analyzed using Western blotting with anti-Flag, anti-GFP, or anti-β-actin antibody.

Article Snippet: The commercial antibodies used in this study were as follows: rabbit anti-Flag antibody and rabbit anti-GFP antibody (HuaAn, China), rabbit anti-IRF3 antibody and rabbit anti-histone H3 antibody (ABclonal, China), rabbit anti-p-IRF3 antibody (CST, USA), rabbit anti-β-tubulin antibody (MBL, Japan), rabbit anti-HA antibody, mouse anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and HRP-conjugated anti-mouse secondary antibody (Sangon Biotech, China), and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, USA).

Techniques: Expressing, Immunoprecipitation, Western Blot, Labeling, Immunostaining, Staining, Transfection, Infection

Journal: Microbiology Spectrum

Article Title: Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation

doi: 10.1128/spectrum.03413-22

Figure Lengend Snippet: aMPV P protein targets IRF3 for inhibiting IFN-β activation in HEK-293T cells. (A) HEK-293T cells cotransfected with IRF3-luc, pRL-TK, expression plasmid P (HA-P), or empty plasmid (HA). At 24 hpt, the cells were transfected with poly (I·C) for 12 h and the luciferase activity measured; the expression of HA-tag protein and β-actin were detected using Western blotting. (B) HEK-293T cells were transfected with expression plasmid P (GFP-P) or empty plasmid (GFP). At 24 hpt, the cells were transfected with poly (I·C) for 12 h, and the expression of IRF3 mRNA was measured using RT-qPCR. (C and D) HEK-293T cells were cotransfected with GFP-P and Flag-IRF3 expression plasmids for 36 h. The cells were processed, immunoprecipitated with anti-GFP, anti-Flag, or anti-β-actin antibody, and analyzed using Western blotting with anti-Flag, anti-GFP, or anti-β-actin antibody. (C and D) HEK-293T cells were cotransfected with GFP-P and Flag-IRF3 expression plasmids for 36 h. The cells were processed, immunoprecipitated with anti-GFP or anti-Flag antibody, and subsequently analyzed using Western blotting with anti-Flag or anti-GFP antibody. (E and F) HEK-293 or A549 cells were cotransfected with GFP-P and/or Flag-IRF3 expression plasmids for 36 h, fixed, and processed. The dual labeling for GFP-P (green) and Flag-IRF3 (red) was observed using immunostaining with anti-Flag antibody. Cell nuclei were stained with DAPI (blue). The yellow observed in the merged images is considered to indicate colocalization areas. Scale bars, 20 μm. Values are presented as the mean ± SD from three independent experiments. Statistical analysis was performed using Student's t test (**, P < 0.01; ****, P < 0.0001).

Article Snippet: The commercial antibodies used in this study were as follows: rabbit anti-Flag antibody and rabbit anti-GFP antibody (HuaAn, China), rabbit anti-IRF3 antibody and rabbit anti-histone H3 antibody (ABclonal, China), rabbit anti-p-IRF3 antibody (CST, USA), rabbit anti-β-tubulin antibody (MBL, Japan), rabbit anti-HA antibody, mouse anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and HRP-conjugated anti-mouse secondary antibody (Sangon Biotech, China), and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, USA).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Labeling, Immunostaining, Staining

Journal: Microbiology Spectrum

Article Title: Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation

doi: 10.1128/spectrum.03413-22

Figure Lengend Snippet: aMPV/C P protein (101 to 200 aa) interacts with IRF3. (A) Schematic representation of aMPV/C P protein (aa 1 to 295) and various truncated P proteins (aa 1 to 200, aa 101 to 295, aa 1 to 100, aa 101 to 200, and aa 201 to 295). (B) HEK-293T cells were cotransfected with GFP-P, GFP-P (1 to 200 aa), or GFP-P (101 to 295 aa) and Flag-IRF3 plasmids for 36 h. The cells were processed, immunoprecipitated with anti-Flag antibody, and analyzed using Western blotting with anti-Flag, anti-GFP antibody, or anti-β-actin. (C) HEK-293T cells were cotransfected with GFP-P, GFP-P (1 to 100 aa), GFP-P (101 to 200 aa), or GFP-P (1 to 200 aa) and Flag-IRF3 plasmids for 36 h. The cells were processed, immunoprecipitated with anti-Flag antibody, and analyzed using Western blotting with anti-Flag, anti-GFP, or anti-β-actin antibody. (D) HEK-293T cells were cotransfected with GFP-P, GFP-P (101 to 200 aa), GFP-P (201 to 295 aa), or GFP-P (101 to 295 aa) and Flag-IRF3 plasmids for 36 h. The cells were processed, immunoprecipitated with anti-Flag antibody, and analyzed using Western blotting with anti-Flag, anti-GFP, or anti-β-actin antibody.

Article Snippet: The commercial antibodies used in this study were as follows: rabbit anti-Flag antibody and rabbit anti-GFP antibody (HuaAn, China), rabbit anti-IRF3 antibody and rabbit anti-histone H3 antibody (ABclonal, China), rabbit anti-p-IRF3 antibody (CST, USA), rabbit anti-β-tubulin antibody (MBL, Japan), rabbit anti-HA antibody, mouse anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and HRP-conjugated anti-mouse secondary antibody (Sangon Biotech, China), and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Microbiology Spectrum

Article Title: Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation

doi: 10.1128/spectrum.03413-22

Figure Lengend Snippet: The effect of P protein on degradation or phosphorylation of IRF3. (A) HEK-293T cells transfected with different doses of expression plasmid P (GFP-P) or GFP. At 36 hpt, the cells were processed, and the expression of IRF3, GFP-tag protein, and β-actin was measured using Western blotting. (B) HEK-293T cells were transfected with expression plasmid P (GFP-P) or empty plasmid (GFP). After 24 h, the cells were transfected with poly (I·C) for 12 h and analyzed using Western blotting for p-IRF3, IRF3, GFP-P, GFP, and β-actin.

Article Snippet: The commercial antibodies used in this study were as follows: rabbit anti-Flag antibody and rabbit anti-GFP antibody (HuaAn, China), rabbit anti-IRF3 antibody and rabbit anti-histone H3 antibody (ABclonal, China), rabbit anti-p-IRF3 antibody (CST, USA), rabbit anti-β-tubulin antibody (MBL, Japan), rabbit anti-HA antibody, mouse anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and HRP-conjugated anti-mouse secondary antibody (Sangon Biotech, China), and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Microbiology Spectrum

Article Title: Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation

doi: 10.1128/spectrum.03413-22

Figure Lengend Snippet: aMPV/C P protein blocks IRF3 nuclear translocation. (A) HEK-293T cells were transfected with expression plasmid P (GFP-P) or empty plasmid (GFP) for 24 h and subsequently transfected with poly (I·C) for 12 h. Cytoplasmic extracts and nuclear extracts were extracted and detected using Western blotting for p-IRF3, IRF3, GFP-P, GFP, and β-actin. (B) A549 cells were transfected and treated with poly (I·C), as described in the legend to panel A. Cells were stained with anti-IRF3 antibody. Cell nuclei were stained with DAPI (blue). Images were obtained using confocal microscopy. Scale bars, 20 μm.

Article Snippet: The commercial antibodies used in this study were as follows: rabbit anti-Flag antibody and rabbit anti-GFP antibody (HuaAn, China), rabbit anti-IRF3 antibody and rabbit anti-histone H3 antibody (ABclonal, China), rabbit anti-p-IRF3 antibody (CST, USA), rabbit anti-β-tubulin antibody (MBL, Japan), rabbit anti-HA antibody, mouse anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and HRP-conjugated anti-mouse secondary antibody (Sangon Biotech, China), and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, USA).

Techniques: Translocation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Confocal Microscopy