monoclonal mouse anti flag tag antibody  (Novus Biologicals)

Journal: PLOS Pathogens

Article Title: RNF20 dynamically regulates RIG-I and MDA5 transcription and degradation via nucleocytoplasmic translocation to balance antiviral signaling

doi: 10.1371/journal.ppat.1013890

Figure Lengend Snippet: (A) IFN-β luciferase reporter assay of HEK293T cells transiently transfected with HA-tagged RIG-I, MDA5, MAVS, STING, or TBK1 (100 ng) together with either RNF20-Flag expression plasmid (100 ng) or an empty control vector, followed by infection with VSV-GFP (MOI = 1.0) for 12h . (B, C) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and HA-tagged RIG-I or MDA5, together with increasing doses of RNF20-Flag expression plasmid (0, 50, 100, 200, 300, or 400 ng). (D) HEK293T cells were transiently co-transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), poly(I:C), and Flag-RIG-I (100 ng), together with RNF20-interfering plasmid. (E, F) HEK293T cells were co-transfected with Flag-RNF20 and either Myc-tagged RIG-I or Myc-tagged MDA5, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. (G, H) Confocal microscopy analysis of HeLa cells infected with NDV for 24 h, followed by immunostaining with anti-RNF20, anti-RIG-I, or anti-MDA5 primary antibodies, and Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Cy3-conjugated goat anti-rat IgG (red) secondary antibodies. (I, J) Schematic representation of RIG-I and MDA5 structures and their truncated mutants. (K) HEK293T cells were co-transfected with Myc-tagged MDA5 or its mutants together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. (L, M) HEK293T cells were co-transfected with Myc-tagged MDA5 or RIG-I (wild-type or dCARD mutants) together with Flag-RNF20, followed by infection with VSV-GFP (MOI = 1.0) for 12h . Cell lysates were immunoprecipitated using an anti-Myc antibody and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD; *** p < 0.001; Scale bars = 10 µm. The results were repeated in three independent experiments with three replicas each. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).

Article Snippet: The antibodies IgG were purchased from the indicated manufacturer’s, including HRP-conjugated anti-mouse or rabbit IgG (Cell Signaling Technologies, 7076 and 7074), Alexa Fluor 488-conjugated Goat anti-rabbit IgG (Sangon Biotech, D110061), Cy3-conjugated Goat anti-mouse IgG (Sangon Biotech, D110088), Anti-Flag tag mouse monoclonal antibody (Sangon Biotech, D191041), Anti-HA tag mouse monoclonal antibody (Abmart, 26D11), Anti-Myc tag mouse monoclonal antibody (Abmart, 19C2), Anti-GFP tag mouse monoclonal antibody (ABclonal,AE012), anti-β-tubulin (Abmart, 2H4), anti-Lamin B1 (Beyotime, AF1408), anti-RNF20 (proteintech, 21625–1-AP), anti-RNF20 (Santa Cruz biotechnology, sc-517358), anti-RIG-I (Cell Signaling Technologies, 3743), anti-MDA5 (Cell Signaling Technologies, 5321), anti-MAVS (Cell Signaling Technologies, 3993), anti-TBK1 (Cell Signaling Technologies, 3504), anti- p -TBK1 (Cell Signaling Technologies, 5483), anti-IRF3 (Cell Signaling Technologies, 11904), anti- p -IRF3 (Cell Signaling Technologies, 4947), anti-STING (Cell Signaling Technologies, 13647S), anti-STAT1 (Sangon Biotech, D120084).

Techniques: Luciferase, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Control, Infection, Immunoprecipitation, Western Blot, Confocal Microscopy, Immunostaining, Virus, shRNA

Journal: PLOS Pathogens

Article Title: RNF20 dynamically regulates RIG-I and MDA5 transcription and degradation via nucleocytoplasmic translocation to balance antiviral signaling

doi: 10.1371/journal.ppat.1013890

Figure Lengend Snippet: (A) Schematic diagram of RNF20 and its deletion mutants. (B) Luciferase reporter assay of HEK293T cells transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and RNF20 (wild-type or mutant) expression plasmids or control vector. (C, D) HEK293T cells were transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and Myc-RIG-I or Myc-MDA5, together with C-terminal truncation mutants of RNF20 (upper panels). Immunoblotting was performed with anti-Myc and anti-β-tubulin antibodies (lower panels). (E) HEK293T cells were co-transfected with Myc-MDA5 and RNF20-HA (wild-type or mutant) expression plasmids. Cell lysates were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting with the indicated antibodies. (F) Co-immunoprecipitation analysis of the polyubiquitination of MDA5 in HEK293T cells transfected with Myc-MDA5, RNF20-Flag (wild-type or mutant), and HA-ubiquitin expression plasmids. (G) Quantification of MDA5 ubiquitination levels in immunoprecipitated samples. (H) Amino acid sequence alignment of the CARD domains of RIG-I and MDA5 showing the conserved “KENW” motif. (I, J) Co-immunoprecipitation analysis of the polyubiquitination of human RIG-I or MDA5 (wild-type or K-to-R mutants) in HEK293T cells transfected with Myc-RIG-I or Myc-MDA5, Flag-RNF20, and HA-ubiquitin expression plasmids. (K, L) Quantification of RIG-I and MDA5 ubiquitination levels in immunoprecipitated samples. (M-P) Immunoblot and quantification analysis of HEK293T cells transiently transfected with RNF20-HA and either RIG-I-Flag or RIG-I-dKENW-Flag, or MDA5-Flag or MDA5-dKENW-Flag, followed by VSV-GFP infection (MOI = 1.0) for 24h . Cell lysates were analyzed by immunoblotting with anti-Flag, anti-HA, and anti-β-tubulin antibodies. Data are shown as mean ± SD from three independent experiments (n = 3). ns: no significance, p < 0.05, * p < 0.01, ** p < 0.001.(Note: pcDNA3.1: empty vector control).

Article Snippet: The antibodies IgG were purchased from the indicated manufacturer’s, including HRP-conjugated anti-mouse or rabbit IgG (Cell Signaling Technologies, 7076 and 7074), Alexa Fluor 488-conjugated Goat anti-rabbit IgG (Sangon Biotech, D110061), Cy3-conjugated Goat anti-mouse IgG (Sangon Biotech, D110088), Anti-Flag tag mouse monoclonal antibody (Sangon Biotech, D191041), Anti-HA tag mouse monoclonal antibody (Abmart, 26D11), Anti-Myc tag mouse monoclonal antibody (Abmart, 19C2), Anti-GFP tag mouse monoclonal antibody (ABclonal,AE012), anti-β-tubulin (Abmart, 2H4), anti-Lamin B1 (Beyotime, AF1408), anti-RNF20 (proteintech, 21625–1-AP), anti-RNF20 (Santa Cruz biotechnology, sc-517358), anti-RIG-I (Cell Signaling Technologies, 3743), anti-MDA5 (Cell Signaling Technologies, 5321), anti-MAVS (Cell Signaling Technologies, 3993), anti-TBK1 (Cell Signaling Technologies, 3504), anti- p -TBK1 (Cell Signaling Technologies, 5483), anti-IRF3 (Cell Signaling Technologies, 11904), anti- p -IRF3 (Cell Signaling Technologies, 4947), anti-STING (Cell Signaling Technologies, 13647S), anti-STAT1 (Sangon Biotech, D120084).

Techniques: Luciferase, Reporter Assay, Transfection, Mutagenesis, Expressing, Control, Plasmid Preparation, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Sequencing, Infection

Journal: PLOS Pathogens

Article Title: RNF20 dynamically regulates RIG-I and MDA5 transcription and degradation via nucleocytoplasmic translocation to balance antiviral signaling

doi: 10.1371/journal.ppat.1013890

Figure Lengend Snippet: (A-C) Confocal microscopy of HeLa cells that were uninfected (UI) or infected with H1N1, NDV, or VSV-GFP for the indicated time points (0, 3, 6, 9, 12, and 24h ) . Cells were stained with an anti-RNF20 primary antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green) or Cy3-conjugated goat anti-rat IgG (red). Scale bars = 10 μm. (D) Immunoblot analysis of RNF20 in cytoplasmic and nuclear fractions of A549 cells infected with VSV-GFP for the indicated time points. (E) Immunoblot analysis of RNF20 in cytoplasmic and nuclear fractions of A549 cells that were UI or infected with VSV-GFP for 24h and then treated with DMSO and KPT330. (F, G) qPCR analysis of RNF20 and IFN-β mRNA levels in A549 cells that were transiently transfected with RNF20 expression or control plasmid, infected with VSV-GFP for 24h and treated with DMSO or KPT330. (H) Co-immunoprecipitation analysis of CRM1-Flag and RNF20-HA in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (I, J) Confocal microscopy of HeLa cells transfected for 24 h with RNF20-dNLS-EGFP or RNF20-dNES-EGFP expression plasmids, followed by infection with NDV for 24 h or left uninfected. Scale bars = 10 μm. (K, L) Luciferase reporter assay of HEK293T cells transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and RNF20 (wild-type or mutant) expression plasmids or control vector. (M, N ) qPCR analysis of IFN-β, OAS1, IL-1β, IL-6, and IL-8 mRNA levels in A549 cells that were transiently transfected with RNF20 (WT) or RNF20-dNLS-expressing plasmid or control plasmid and infected with VSV-GFP for 24h. n = 3; Data were represented by the mean ± SD with corresponding significance. * p < 0.05, ** p < 0.01, *** p < 0.001. (Note: UI denotes uninfected virus; pcDNA3.1: empty vector control; shNC: non-targeting shRNA control).

Article Snippet: The antibodies IgG were purchased from the indicated manufacturer’s, including HRP-conjugated anti-mouse or rabbit IgG (Cell Signaling Technologies, 7076 and 7074), Alexa Fluor 488-conjugated Goat anti-rabbit IgG (Sangon Biotech, D110061), Cy3-conjugated Goat anti-mouse IgG (Sangon Biotech, D110088), Anti-Flag tag mouse monoclonal antibody (Sangon Biotech, D191041), Anti-HA tag mouse monoclonal antibody (Abmart, 26D11), Anti-Myc tag mouse monoclonal antibody (Abmart, 19C2), Anti-GFP tag mouse monoclonal antibody (ABclonal,AE012), anti-β-tubulin (Abmart, 2H4), anti-Lamin B1 (Beyotime, AF1408), anti-RNF20 (proteintech, 21625–1-AP), anti-RNF20 (Santa Cruz biotechnology, sc-517358), anti-RIG-I (Cell Signaling Technologies, 3743), anti-MDA5 (Cell Signaling Technologies, 5321), anti-MAVS (Cell Signaling Technologies, 3993), anti-TBK1 (Cell Signaling Technologies, 3504), anti- p -TBK1 (Cell Signaling Technologies, 5483), anti-IRF3 (Cell Signaling Technologies, 11904), anti- p -IRF3 (Cell Signaling Technologies, 4947), anti-STING (Cell Signaling Technologies, 13647S), anti-STAT1 (Sangon Biotech, D120084).

Techniques: Confocal Microscopy, Infection, Staining, Western Blot, Transfection, Expressing, Control, Plasmid Preparation, Immunoprecipitation, Luciferase, Reporter Assay, Mutagenesis, Virus, shRNA