Journal: PLoS ONE

Article Title: Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1

doi: 10.1371/journal.pone.0173454

Figure Lengend Snippet: a) Glycosylation analysis of purified gp41. The protein was denatured and left untreated or incubated with Endoglycosidase H (EndoH) or with PNGase F (PNGase) as indicated. The exclusive sensitivity to PNGase F digestion suggests that the protein is properly post-translationally modified with N-linked sugar residues. b) Analysis of the purified protein using native PAGE, The migration of gp41 at ~160 kDa suggests a trimeric state of the protein. c) Binding of different monoclonal antibodies to recombinant gp41. Binding was measured in a capture ELISA at 4 or 37°C incubation temperature with indicated antibodies coated to the plate. 2nd, secondary anti HIS-HRP probe only, human, anti-gp120 control antibody, mouse, anti gp120 control antibody. For details on the monoclonal antibodies, please see . Different levels of binding and a temperature sensitive recognition pattern was observed for selected antibodies. d) Results of surface plasmon resonance studies using recombinant gp41 and various monoclonal antibodies. Single cycle kinetics were determined by capturing the indicated anti-gp41 antibodies to the chip via covalently linked anti-mouse or anti human IgG, followed by five injections of gp41 at increasing concentrations. Data shown (ΔRU) represent double referenced sensograms with relative signal intensities obtained by subtraction of flow cell 2 (gp41 antibody) and flow cell 1 (no antibody). Measurement curves (black) and fitted curves (red) are indicated.

Article Snippet: Affinity measurements were performed on a Biacore X100 device (GE Healthcare, Germany) at 37°C using the human or mouse antibody capture kit and CM5 sensor chips according to manufacturer’s recommendations (GE Healthcare, Germany).

Techniques: Purification, Incubation, Modification, Clear Native PAGE, Migration, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, SPR Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: A mutation in NLRC4 induces overproduction of IL-1β through activation of caspase-1. (a) Flag-tagged wild-type, mutant NLRC4 expression vectors or empty vector (EV) were transfected into 293T cells. 2 d later, after the cell lysates were subjected to SDS-PAGE, Western blotting was performed with an anti-Flag or anti-actin antibody. Red triangle indicates NLRC4. The data in the figure are representative of three independent experiments. (b) Cell lysates from 293T cells transfected with empty vector (EV), flag-tagged wild-type, or mutant NLRC4 were subjected to Blue Native PAGE and the expression of the tagged proteins or actin was evaluated by Western blotting with an anti-Flag or actin antibody, respectively. The data in the figure are representative of three independent experiments. (c) 293T cells were transfected with a CASP1 expression vector together with empty vector (EV), an expression vector for wild-type or mutant NLRC4 . 24 h later, the expression of procaspase-1, cleaved active caspase-1 (10 kD), and actin was evaluated by Western blotting. Red triangles indicate procaspase-1, cleaved active caspase-1, and actin. The data in the figure are representative of five independent experiments. (d) 293T cells were transfected with expression vectors for CASP1 and IL1B together with empty vector (EV), an expression vector for wild-type or mutant NLRC4 . 24 h later, the concentration of IL-1β in the supernatant was evaluated by ELISA. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (e) Peripheral blood mononuclear cells were transfected with three concentrations of Prgl and cultured for 48 h. The concentrations of IL-1β in the supernatants were measured by ELISA. The data shown are means ± SD (**, P < 0.01). The data in the figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Mutagenesis, Activation Assay, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, Blue Native PAGE, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: BM-derived macrophages that overexpress mutant Nlrc4 secrete IL-1β. BM-derived macrophages (BMMC) infected with control retrovirus, retroviruses encoding wild-type, or mutant Nlrc4 were stimulated with 10 µg/ml LPS for 1 d in the absence (black) or presence (gray) of a caspase-1 inhibitor. As the control, unstimulated BMMCs were used (white). The concentrations of IL-1β and IL-6 in the supernatant were measured by ELISA. The data shown are means ± SD (*, P < 0.05; n = 5). The data shown in this figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Derivative Assay, Mutagenesis, Infection, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: Cytokine production in mu-Nlrc4 mice or mice reconstituted with mutant Nlrc4 transduced BM cells. (a) Splenocytes from wild-type or mu-Nlrc4 mice were stimulated with LPS for 24 h, and the concentration of IL-1β in each supernatant was evaluated by ELISA. The data shown are means ± SD (**, P < 0.01). N.D.: not detected. The data in the figure are representative of three independent experiments. (b) The concentrations of IL-1β, IL-17A, and G-CSF in the serum of wild-type (white) and mu-Nlrc4 (black) mice at the age of 8 wk were evaluated by ELISA. For all panels, the data shown are means ± SD (**, P < 0.01; n = 5). N.D.: not detected. The data in the figure are representative of three independent experiments. (c) Intraperitoneal cells or splenocytes from C57BL/6 mice transplanted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus were evaluated for the number of Gr1 + CD11b + cells 2 mo after the transplantation. The data shown are means ± SD (*, P < 0.05; n = 5 in each group). The data in the figure are representative of three independent experiments. (d) Irradiated C57BL/6 mice were reconstituted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus. Serum IL-1β, IL-17A, and G-CSF were evaluated by ELISA 6 wk after BM transplantation. For all panels, the data shown are means ± SD (**, P < 0.01; n = 5). N.D.: not detected. The data in the figure are representative of two independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Mutagenesis, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, Control, Virus, Transplantation Assay, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: Cold exposure of mu-Nlrc4 mice increases autoinflammation. (a) The footpads of C57BL/6 (wild type; white) and mu-Nlrc4 (black) mice were exposed to 4°C for 5 min. The thickness of each footpad was measured before and after exposure to the cold stimulus. The data are shown as the ratio of the thickness after exposure to the thickness before exposure. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (b) Irradiated C57BL/6 mice were reconstituted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus. The footpads were exposed to 4°C for 5 min. The thickness of each footpad was measured before and after exposure to the cold stimulus. The data are shown as the ratio of the thickness after exposure to the thickness before exposure. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (c) The entire bodies of control C57BL/6 and mu-Nlrc4 mice were exposed to 4°C for 1 min and kept for 3 min at room temperature, and then the serum concentrations of IL-1β were measured. The data are shown as means ± SD. N.D., not detected. (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (d) MC/9 cells infected with control retrovirus (white), retroviruses encoding wild-type (black), or mutant Nlrc4 (gray) were cultured at 32 or 37°C for 48 h. 293T cells were transfected with control vector (white), vectors encoding wild-type (black), or mutant NLRC4 (gray) and 48 h later, cells were washed and cultured at 32 or 37°C for 6 h. The levels of IL-1β in the supernatants were evaluated by ELISA. The data are shown as the mean ratio of the IL-1β level in cells cultured at 32°C to the level in cells cultured at 37°C. The data are shown as means ± SD (*, P < 0.05). The data in the figure are representative of three independent experiments. (e) Peripheral mononuclear cells from an FCAS patient or a healthy control were cultured at 32°C for 48 h in the presence (black) or absence (white) of a caspase inhibitor or cultured at 37°C for 48 h. The level of IL-1β in the supernatant was evaluated by ELISA. The data are shown as the mean ratio of the IL-1β level in cells cultured at 32°C to the level in cells cultured at 37°C. The data are shown as means ± SD (**, P < 0.01). The data in the figure are representative of two independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Irradiation, Infection, Control, Virus, Mutagenesis, Cell Culture, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: The accumulation of neutrophils in mu-Nlrc4 mice depends on IL-1β and IL-17A. (a) Spleen cells from C57BL/6 or mu-Nlrc4 mice at the age of 12 wk were stimulated with 25 ng/ml PMA and 1 µg/ml ionomycin in the presence of 2 µM monensin for 5 h and then stained with anti–TCR-β, anti–TCR-γδ, anti-B220, anti-CD11c, anti-NK1.1, anti-Gr1, and anti-CD11b antibodies. The cells were fixed and stained with anti–IL-17A antibody. The expression of IL-17A was analyzed by flow cytometry. The number in each rectangle indicates the percentage of IL-17A–positive cells in the lineage-negative fraction. The data in the figure are representative of five independent experiments. (b) mu-Nlrc4 mice at the age of 12 wk were injected with control IgG, anti-CD4, anti-CD8, anti-Thy1.2, anti-Gr1, or anti–IL-1β antibody five times at 3-d intervals. The serum IL-17A levels 2 d after final antibody treatment was measured by ELISA. Control sera from C57BL/6 (wild type) mice was used. The data shown are means ± SD (**, P < 0.01; n = 5 for all). The data in the figure are representative of three independent experiments. (c) mu-Nlrc4 mice at the age of 12 wk were injected twice with control rat IgG, anti-CD4, anti-CD8, or anti-Thy1.2 at 3 d intervals. The spleen cells from mice 1 d after the final injection were stained with anti-CD4 and anti–TCR-β (for anti-CD8–treated mice), anti-CD8 and anti–TCR-β (for anti-CD4–treated mice), or anti-CD4, anti-CD8, and anti–TCR-β antibodies (for anti-Thy1.2–treated mice). The data in the figure are representative of two independent experiments. (d) mu-Nlrc4 mice at the age of 12 wk were injected with control IgG, anti–IL-1β, anti–IL-17A, or anti–IL-1β, and anti–IL-17A antibody five times at 3-d intervals. The number of Gr1 + CD11b + cells in the spleen 2 d after the final antibody treatment was evaluated by flow cytometry. The data shown are means ± SD (*, P < 0.05; **, P < 0.01; n = 5 for all). The data in the figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Staining, Expressing, Flow Cytometry, Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: Emerging Microbes & Infections

Article Title: Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway

doi: 10.1080/22221751.2020.1745094

Figure Lengend Snippet: NS4A interacts with the SH2 and TAD domain of STAT1 or STAT2 to impair their phosphorylation. (A) In vitro kinase assay of STAT1 phosphorylated at Tyr701 (Y701) and STAT2 phosphorylated at Tyr690 (Y690). The peptide of STAT1 or STAT2 and their kinase JAK1 or TYK2 were introduced into a mixture containing Flag-NS4A. (B) Schematic representation of STAT1 or STAT2 (top). IB analysis of HEK293 T cells co-transfected for 48 h with Flag-NS4A and HA-wild-type STAT1 or STAT2, or their mutants, assessed with whole-cell lysates (10% input) or IP with anti-HA antibody (bottom). The numbers in A means the relative intensity of the p-STAT1/ STAT1 or p-STAT2/STAT2 detected by Western blotting. Data are representative of three independent experiments.

Article Snippet: Rabbit anti-Flag, HA, STAT1, phospho-STAT1 (Tyr701), STAT2, phospho-STAT2 (Tyr690), IRF-9, Ub and phospho-Jak family antibody sampler kit were all purchased from Cell Signalling Technology.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Transfection, Western Blot

Journal: Emerging Microbes & Infections

Article Title: Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway

doi: 10.1080/22221751.2020.1745094

Figure Lengend Snippet: NS4 inhibits the signalling pathway induced by type I IFN. (A) Tyrosine phosphorylation of STATs in TBEV or VSV-infected T98G cells. T98G cells at 1 × 10 6 infected by TBEV (MOI = 1) or VSV (MOI = 0.1) at various time points were collected and lysed, and the whole lysates were prepared. Western blotting was performed to determine the levels of total and tyrosine-phosphorylated (p-) STAT1 (left, top) and STAT2 (left, bottom) during TBEV or VSV infection. GAPDH was used as a loading control. Histogram (right) representing a densitometric analysis performed to quantify the relative intensity of the indicated bands detected by Western blotting. (B) NS4A suppressed the ISRE reporter activity in IFNα or IFNβ stimulation. HEK293 T (5 × 10 4 ) was transfected with Flag-NS4A (200 ng), luciferase reporter plasmid for ISRE (100 ng) and pRL-TK (5 ng), then stimulated with INF-α or IFN-β (50 ng/mL). The reporter activity was detected as Fig.1B. (C) Effect of NS4A on INF-α or IFN-β induced transcription of ISG15, ISG54 and ISG56 genes. HEK293 T (1 × 10 6 ) was transfected with Flag-NS4A (2 μg) and then stimulated with INF-α or IFN-β (50 ng/mL). Levels of ISG15, ISG54, and ISG56 mRNAs were measured by real-time RT-PCR analysis, normalized to the cellular β-actin mRNA, and set in relation to mRNA levels of mock-infected cells. (D) Microscopy imaging of vector or NS4A transfected HEK293 T uninfected or infected with HSV-1-GFP (0.1 M.O.I.) or VSV-GFP (0.1 M.O.I.) for 24 h. Scale bar, 20 μm. (Right) Quantitative measurements of GFP in HEK293T cells infected with HSV-1-GFP or VSV-GFP. Student’s t -test was used for estimation of statistical significance. *, P < 0.05; **, P < 0.01 and ***, P < 0.001. Data are from three independent experiments. Mean values and standard deviations from three independent experiments are shown.

Article Snippet: Rabbit anti-Flag, HA, STAT1, phospho-STAT1 (Tyr701), STAT2, phospho-STAT2 (Tyr690), IRF-9, Ub and phospho-Jak family antibody sampler kit were all purchased from Cell Signalling Technology.

Techniques: Phospho-proteomics, Infection, Western Blot, Control, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Quantitative RT-PCR, Microscopy, Imaging

Journal: Emerging Microbes & Infections

Article Title: Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway

doi: 10.1080/22221751.2020.1745094

Figure Lengend Snippet: NS4A inhibits the phosphorylation and dimerization of STAT1/STAT2. (A&B) HEK293 T at 1 × 10 6 mL was transfected with NS4A for 24 h then infected with mock or SeV for 8 h. (A)The total cell lysates were prepared and the tyrosine-phosphorylated STAT1 or STAT2 and phosphorylated JAK or TYK2 were determined by Western blotting. Meanwhile, the total amounts of STAT1, STAT2, JAK, TYK and NS4A were determined. ACTIN was used as a loading control. (B) Native PAGE analysis of STAT1/2 in dimer or monomer form. (C&D) HEK293 T at 1 × 10 6 mL was transfected with NS4A for 24 h then treated with INF-α or IFN-β (50 ng/mL). The total cell lysates were prepared at indicated time. (C) The phosphorylated STAT1 or STAT2 and total STAT1, STAT2 and NS4A were determined by Western blotting. ACTIN was used as a loading control. The numbers in A&C means the relative intensity of the p-STAT1/ STAT1 or p-STAT2/STAT2 detected by Western blotting. (D) The dimer of STAT1/2 was analysed by Native PAGE. Data are representative of three independent experiments.

Article Snippet: Rabbit anti-Flag, HA, STAT1, phospho-STAT1 (Tyr701), STAT2, phospho-STAT2 (Tyr690), IRF-9, Ub and phospho-Jak family antibody sampler kit were all purchased from Cell Signalling Technology.

Techniques: Phospho-proteomics, Transfection, Infection, Western Blot, Control, Clear Native PAGE

Journal: Emerging Microbes & Infections

Article Title: Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway

doi: 10.1080/22221751.2020.1745094

Figure Lengend Snippet: NS4A suppresses the formation of ISGF3. (A) The interaction of NS4A with JAK1, TYK2, STAT1, STAT2 or IRF9. (A) The interaction of NS4A with JAK1, TYK2, STAT1, STAT2 or IRF9. IP and IB analyses of lysates of HEK293 T cells (1 × 10 6 ) overexpressing plasmids encoding Myc-tagged NS4A or Flag-JAK1, Flag-TYK2, Flag-STAT1, Flag-STAT2 or Flag-IRF9. (B) The effect of NS4A present on the interaction between the pairings of ISGF3 complex. IP and IB analyses the association of STAT1, STAT2 and IRF9 in the presence or absence of Flag-NS4A. (C) IP and IB analyses the interaction of STAT1 and STAT2 when Flag-NS4A gradient overexpression. (D) IP and IB analyses the formation of ISGF3 in the presence or absence of Flag-NS4A. (E) IP and IB analyses the interaction of TYK2 with STAT1 or the interaction of JAK1 with STAT2 in the presence or absence of Flag-NS4A. Data are representative of three independent experiments.

Article Snippet: Rabbit anti-Flag, HA, STAT1, phospho-STAT1 (Tyr701), STAT2, phospho-STAT2 (Tyr690), IRF-9, Ub and phospho-Jak family antibody sampler kit were all purchased from Cell Signalling Technology.

Techniques: Over Expression

Journal: Emerging Microbes & Infections

Article Title: Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway

doi: 10.1080/22221751.2020.1745094

Figure Lengend Snippet: The ubiquitination of NS4A at Lys132 is important for the interaction of NS4A and STAT1. (A) HEK293 T cells (1 × 10 6 ) were transfected with the indicated plasmids. 24 h after transfection, ubiquitination assays and immunoblotting analysis were performed with the indicated antibodies. (B) Effect of K132R mutant on ubiquitination of NS4A. The experiments were performed as in A except that the indicated plasmids were used. (C) Effect of K132R mutant on the interaction of NS4A with STAT1. HEK293 T cells (1 × 10 6 ) transfected with the indicated plasmids (1 μg each) for 36 h followed by co-immunoprecipitation experiments and immunoblotting analysis with the indicated antibodies. (D) Effect of K132R mutant on the dimer of STAT1 and STAT2. The cells were performed as in C. The dimer of STAT1/2 was analysed by Native PAGE. (E) Effect of NS4A peptide on the interaction of STAT1 and STAT2. HEK293 T cells (1 × 10 6 ) were transfected with Flag-STAT1 and HA-STAT2. 48 h after transfection, the cells were lysed and then added the different peptides of NS4A, IP analyses the interaction of STAT1 with STAT2. (F&G) Immunoblot analysis of the ubiquitination of NS4A in T98G cells co-transfected with HA-Ub, HA-Ub-K27R (F) or HA-Ub-K27O (G). Data are representative of three independent experiments.

Article Snippet: Rabbit anti-Flag, HA, STAT1, phospho-STAT1 (Tyr701), STAT2, phospho-STAT2 (Tyr690), IRF-9, Ub and phospho-Jak family antibody sampler kit were all purchased from Cell Signalling Technology.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Mutagenesis, Immunoprecipitation, Clear Native PAGE