Journal: Nature immunology

Article Title: Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity

doi: 10.1038/ni.3542

Figure Lengend Snippet: (a) Immunoassay of the interaction of MAVS with PCBP2 in lysates of 293T cells transfected to express FLAG-tagged MAVS and plasmids encoding various GST-tagged constructs of PCBP2 (above lanes), assessed by co-immunoprecipitation with anti-FLAG and immunoblot analysis with anti-GST (input, as in Fig. 4a throughout). (b) In vitro precipitation assay (as in Fig. 6j) of cells as in a: arrowhead, MAVS. (c) GST-precipitation and immunoblot analysis of the interaction of PCBP2 with MAVS (top) or EPRS (bottom) in 293T cells transfected to express various combinations (above lanes) of FLAG-tagged MAVS and GST-tagged PCBP2 plus increasing amounts (wedges) of Strep-tagged EPRS, probed with anti-FLAG or anti-Strep. (d) GST-precipitation assay (as in c) of interactions between PCBP2 and Itch (top) and EPRS (bottom). (e) Immunoassay of the endogenous interactions between PCBP2 and EPRS or MAVS in lysates of PR8-infected RAW264.7 cells, assessed by immunoprecipitation with anti-PCBP2 (for endogenous PCBP2) and immunoblot analysis with anti-EPRS (top) or anti-MAVS (bottom). (f,g) Immunoblot analysis of exogenous MAVS (f) or endogenous MAVS (g) immunoprecipitated from lysates of 293T cells treated with the proteasome inhibitor MG-132 and then transfected to express various combinations (above lanes) of Strep-tagged EPRS, GST-tagged PCBP2, V5-tagged Itch, FLAG-tagged MAVS (f only) and hemagglutinin (HA)-tagged ubiquitin, probed with antibody to Lys48 (K48)-linked ubiquitin (K48-Ub) and other antibodies (right margin). (h,i) Immunoblot analysis (top) of exogenous MAVS (h) or endogenous MAVS (i) in 293T cells transfected to express FLAG-tagged PCBP2, FLAG-tagged MAVS (h only) and increasing amounts of Strep-tagged EPRS. Below, MAVS band intensity, normalized to that of actin (numbers above bars indicate specific intensity). (j) In vitro assay of the ubiquitination of purified Strep-tagged MAVS after incubation with ubiquitin, E1, E2, and a combination of purified Strep-tagged EPRS, Strep-tagged PCBP2 and V5-tagged Itch, assessed by immunoblot analysis with anti-ubiquitin. Data are representative of three experiments with similar results, with three independent biological replicates.

Article Snippet: Other reagents and materials included MG-132 (Sigma), puromycin (Gibco-BRL), poly(I:C) (InvivoGen), IFN-γ(R&D Systems), digitonin (Sigma), protein A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology), Glutathione Sepharose 4 Fast Flow (17–5132-01, GE Healthcare), anti-FLAG M2 affinity gel (A2220, Sigma), Strep-Tactin Sepharose (2–1201-002, IBA), Ni-NTA agarose (30230, Qiagen), GFP-trap (gta-20, ChromoTek), and a Superdex 200 10/300 GL column (GE Healthcare).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, In Vitro, Infection, Purification, Incubation

Journal: Nature immunology

Article Title: Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity

doi: 10.1038/ni.3542

Figure Lengend Snippet: (a) Expression of genes encoding MSC components (right margin) in bronchial epithelial cells (two replicates; one per column) at various times after infection with PR8 (above columns), showing genes upregulated (yellow) or downregulated (blue) over 1.5-fold relative to their expression before infection (key). (b) Luciferase activity of 293T cells transfected with the control TK-Renilla plasmid and a firefly luciferase reporter plasmid containing the IFNB promoter, plus empty vector (EV) or vector encoding various MSC components (horizontal axis), together with plasmid encoding the amino-terminal domain of RIG-I (N-RIG-I); results are presented relative to those of the renilla luciferase control. Below, immunoblot analysis of Strep-tagged MSC proteins and FLAG-tagged N-RIG-I in total lysates of the cells above. (c,d) Viral replication in RAW264.7 cells transfected with EPRS-specific siRNA (siEPRS) or control (non-targeting) siRNA (siCtrl) (key) and infected with PR8-GFP (multiplicity of infection (MOI) = 1) (top row) or VSV-GFP (MOI = 0.5) (bottom row), assessed by fluorescence microscopy (c) and fluorescence absorbance and plaque assay (d) at 24 h after infection. PFU, plaque-forming units. (e,f) Concentration of IFN-β (e) or IL-6 (f) in supernatants of RAW264.7 cells transfected with siRNA as in c,d (key) and infected with PR8-GFP, VSV-GFP or HSV-GFP (MOI = 1) or treated with poly(I:C) (80 µg) (above plots). (g) Immunoblot analysis of phosphorylated (p-) and total (inactive) IRF3 and STAT1, and of EPRS and actin (loading control), in PR8-GFP-infected RAW264.7 cells expressing EPRS-specific (shEPRS) or non-targeting control (shCtrl) short hairpin RNA. (h–j) Fluorescence microscopy (h), fluorescence absorbance and plaque assay (i), and secretion of IFN-β or IL-6 (j) of RAW264.7 cells transfected with empty vector (Ctrl) or vector encoding EPRS and infected with PR8-GFP. Scale bars (c,h), 100 µm. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test). Data are representative of one experiment (a) or three experiments with similar results (b–j), with at least three (b–f,h–j) or two (g) independent biological replicates (mean and s.d. of triplicates in b,d,e,f,i,j).

Article Snippet: Other reagents and materials included MG-132 (Sigma), puromycin (Gibco-BRL), poly(I:C) (InvivoGen), IFN-γ(R&D Systems), digitonin (Sigma), protein A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology), Glutathione Sepharose 4 Fast Flow (17–5132-01, GE Healthcare), anti-FLAG M2 affinity gel (A2220, Sigma), Strep-Tactin Sepharose (2–1201-002, IBA), Ni-NTA agarose (30230, Qiagen), GFP-trap (gta-20, ChromoTek), and a Superdex 200 10/300 GL column (GE Healthcare).

Techniques: Expressing, Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Fluorescence, Microscopy, Plaque Assay, Concentration Assay, shRNA

Journal: Nature immunology

Article Title: Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity

doi: 10.1038/ni.3542

Figure Lengend Snippet: (a) Plaque assay of viral titers in Eprs+/+ and Eprs+/− BMDMs infected with VSV-GFP (MOI = 5) (top) or PR8-GFP (MOI = 3) (bottom). (b,c) Concentration of IFN-β and IL-6 in culture supernatants of cell as as in a after viral infection as in a (b) or treatment with poly(I:C) (40 µg) (c). (d) Expression of Ifnb, Il6 and other genes encoding interferon-related antiviral products (vertical axes) in Eprs+/+ and Eprs+/− mouse-derived BMDMs at 12 h after infection with VSV-GFP. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test). Data are representative three experiments with similar results, with three (a–c) or two (d) independent biological replicates (mean and s.d. of triplicates).

Article Snippet: Other reagents and materials included MG-132 (Sigma), puromycin (Gibco-BRL), poly(I:C) (InvivoGen), IFN-γ(R&D Systems), digitonin (Sigma), protein A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology), Glutathione Sepharose 4 Fast Flow (17–5132-01, GE Healthcare), anti-FLAG M2 affinity gel (A2220, Sigma), Strep-Tactin Sepharose (2–1201-002, IBA), Ni-NTA agarose (30230, Qiagen), GFP-trap (gta-20, ChromoTek), and a Superdex 200 10/300 GL column (GE Healthcare).

Techniques: Plaque Assay, Infection, Concentration Assay, Expressing, Derivative Assay

Journal: Nature immunology

Article Title: Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity

doi: 10.1038/ni.3542

Figure Lengend Snippet: (a,b) Luciferase assay (as in Fig. 1b) of IFNB promoter activation in 293T cells at 24 h after transfection (above plots) to express N-RIG-I, MDA5, poly(I:C) and MAVS (a) or TRAF3, TBK1 and IRF7 (b), and with increasing concentrations (0, 200 or 800 ng; wedges) of plasmid encoding FLAG-tagged EPRS (horizontal axes). (c) Luciferase assay (as in Fig. 1b) of IFNB promoter activation in Toll-like-receptor-3-expressing 293T cells (293T(TLR3)) transfected for 24 h with increasing concentrations (0, 50, 200 or 800 ng; wedges) of plasmid encoding FLAG-tagged EPRS (horizontal axis), followed by stimulation for 12 h with poly(I:C) (30 µg). (d) Silver staining (top) of Strep-EPRS complexes purified from 293T cells 24 h after transfection with a plasmid encoding Strep-EPRS, followed by infection for 6 h with PR8-GFP (MOI = 5): left margin, size in kilodaltons (kDa); right margin, Strep-tagged full-length EPRS (170 kDa); *, PCBP2 (38 kDa). Below, sequences of peptides identified by mass spectrometry. (e,f) Immunoassay of the interaction between EPRS and PCBP2 in PR8-infected RAW264.7 cells (e) and U937 cells (f), assessed by immunoprecipitation with anti-EPRS, followed by immunoblot analysis with anti-PCBP2 (input, as in Fig. 4a). (g) Confocal microcopy of endogenous EPRS (red) and PCBP2 (green) in HeLa cells at various times (left margin) after infection with PR8 (MOI = 5). Scale bars, 10 µm. NS, not significant (P > 0.05); *P < 0.01 and **P < 0.001 (Student’s t-test). Data are representative of three experiments with similar results, with three independent biological replicates (a–c,e–g; mean and s.d. of triplicates in a–c) or one experiment (d).

Article Snippet: Other reagents and materials included MG-132 (Sigma), puromycin (Gibco-BRL), poly(I:C) (InvivoGen), IFN-γ(R&D Systems), digitonin (Sigma), protein A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology), Glutathione Sepharose 4 Fast Flow (17–5132-01, GE Healthcare), anti-FLAG M2 affinity gel (A2220, Sigma), Strep-Tactin Sepharose (2–1201-002, IBA), Ni-NTA agarose (30230, Qiagen), GFP-trap (gta-20, ChromoTek), and a Superdex 200 10/300 GL column (GE Healthcare).

Techniques: Luciferase, Activation Assay, Transfection, Plasmid Preparation, Expressing, Silver Staining, Purification, Infection, Mass Spectrometry, Immunoprecipitation, Western Blot