Journal: Cell Communication and Signaling

Article Title: Intracellular signaling prevents effective blockade of oncogenic gp130 mutants by neutralizing antibodies

doi: 10.1186/1478-811x-12-14

Figure Lengend Snippet: Figure 3 Functional analysis of individual cytoplasmic Tyr-residues of CAgp130. (A) Schematic overview of add-back mutants of CAgp130. EP: extracellular part with depicted del(Y186-Y190), TD: transmembrane domain, CP: cytoplasmic part. HEK293 cells stably expressing IL-6Rα were transiently transfected with WTgp130-YFP, CAgp130-YFP, CAgp130-6F-YFP or YFP-tagged add-back mutants of CAgp130. (B) Overall receptor expression was assessed by FACS analysis of the fluorescent tag (right panel). Surface receptor expression was verified using the gp130 Abs B-P8 and B-R3 and an Alexa633 labeled secondary Ab (left panel). (C) and (D) Cells were stimulated with 200 U/ml IL-6 for the indicated periods of time or left untreated. TCLs were analyzed by immunoblotting. (C) Activation of the JAK/Stat pathway was verified by Abs against pStat3(Y705), pStat1(Y701), Stat3, Stat1 and actin as loading control. (D) JAK/Erk pathway activation was assessed using Abs against pSHP2, pErk1/2, SHP2 and Erk1/2.

Article Snippet: Lysates were subjected to SDS-PAGE, Western Blotting and immunodetection using Abs against pStat3(Y705), pStat3(S727), pStat1 (Y701), pSHP2(Tyr542), pErk1/2(Thr202/Tyr204), Stat1, SHP2, Erk1/2 (Cell signaling, USA), Stat3 (BD transduction laboratories), gp130, SOCS3, dynamin, actin (Santa Cruz Biotechnology, USA), GFP (Rockland, USA) and horseradish-peroxidase conjugated secondary antibodies (DAKO, Denmark).

Techniques: Functional Assay, Stable Transfection, Expressing, Transfection, Labeling, Western Blot, Activation Assay, Control

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: KLC1 interaction with DENV NS1 in infected C6/36 cells. ( a ) Colocalization assays of KLC1 and DENV NS1 (Pearson=0.52). ( b ) PLA of KLC1 and DENV NS1. Mock-infected cells treated with anti-KLC1 ( mock ) and infected cells with primary anti-KLC1 antibody omitted ( control ) were included as negative controls. Crop in (a) and (b); enlargement of the cells enclosed in the yellow square. ( c ) Immunoprecipitation assays. Cell lysates were immunoprecipitated with anti-NS1 antibodies bound to Sepharose beads. The presence of DENV KLC1 in the immunoprecipitant was revealed by western blot. Changes in protein migration observed in the immunoprecipitate are due to elution buffer composition and were consistently observed in experimental replicas. In all cases, mock and infected cells were harvested at 24 hpi. C6/36 average cell diameter=12.5 µm. Bar=5 µm. Representative images of at least three independent experiments are shown.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Infection, Control, Immunoprecipitation, Western Blot, Migration

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: In silico modelling of mosquito KLC1. ( a ) AlphaFold 3.0 modelling of the KLC1 of A. albopictus . TRP domain shown in blue. (b) Superimposition of the TPR cargo binding domain of mosquito (blue) and human (red) KLC1. ( c ) Modelled interaction of the TPR (blue) and NS1 dimers (orange). pTM: predicted template modelling. ipTM: interface predicted template modelling. RMSD: root mean square deviation. Circular arrow=180° image rotation.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: In Silico, Binding Assay

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Immunoelectron microscopy of C6/36 cells infected with DENV. KLC1 was labelled with 30 nm (red arrowheads), and NS1 with 15 nm (green arrowheads) gold particles. (a) Mock-infected cells; (b) and (c) infected cells. Cells were fixed at 24 hpi. KLC1 and NS1 were found to colocalize around vacuole-like structures. N=cell nuclei. Blue arrowheads=lipid cores.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Immuno-Electron Microscopy, Infection

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: KLC1 expression levels in DENV-infected cells. Cell lysates of mock- and DENV-infected cells were analysed by western blot. Relative KLC1 protein levels are expressed as a ratio to β -actin used as load control. Representative images of three independent experiments are shown. * P ≤0.005.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Expressing, Infection, Western Blot, Control

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Effect on DENV replicative cycle of silencing the expression of KLC1. ( a ) Transfection of C6/36 cells with siRNA specific for KLC1 or an NR siRNA. Cells were lysed 24 hpt, and the level of protein expression was analysed by western blot. Relative KLC1 protein levels are expressed as a ratio to GAPDH. ( b ) Expression levels of NS1 in DENV-infected cells silenced or not for KLC1 and lysed 24 hpi. NS1 levels are expressed as a ratio to GAPDH. ( c ) Amount of secreted NS1 in cells silenced or not for the expression of KLC1. Secreted NS1 was measured by an in-house ELISA, expressed as a percentage of the control condition, taken as 100%. ( d ) Virus genome levels. RNA levels were determined by qRT-PCR. ( e ) Virus yield. Virus progeny was determined by plaque assay. Cells were lysed and supernatants collected at 24 hpi. * P ≤0.005. **** P ≤0.001. n ≥3.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Expressing, Transfection, Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Control, Virus, Quantitative RT-PCR, Plaque Assay

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Effect on DENV RC of cells silenced for KLC1. Cells transfected with an NR siRNA. Cells transfected with a KLC1-specific siRNA. Cells were fixed at 24 hpi. Bar=5 µm. Representative images of three independent experiments are shown.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Transfection

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Effect of the KINTAG peptide on the replication of DENV. ( a ) Fluorescence intensity of NS1 in cells transfected with KINTAG peptide fixed at 24 hpi. ( b ) Expression levels of NS1 in DENV-infected cells with KINTAG peptide 24 hpi. NS1 levels are expressed as a ratio to tubulin. ( c ) Amount of secreted NS1 in cells with KINTAG peptide. Secreted NS1 was measured by an in-house ELISA, expressed as a percentage of the control condition, taken as 100%. (d) Virus genome levels. RNA levels were determined by qRT-PCR. ( e ) Virus yield. Virus progeny was determined by plaque assay. Cells were lysed and supernatants collected at 24 hpi. ( f ) Comparison between 6SWU crystal structure (purple) and C6/36’s KLC1 TPR and KINTAG peptide interaction predicted by AlphaFold (blue); KINTAG peptide is shown in green. Bar=10 µm. * P ≤0.005. **** P ≤0.001. n ≥3.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Fluorescence, Transfection, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Control, Virus, Quantitative RT-PCR, Plaque Assay, Comparison

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Effect of KLC1 silencing or function disruption on LD homeostasis. ( a ) Mock-infected cells silenced (siRNA) or not (NR) for KLC1 expression. ( b ) DENV-infected cells silenced or not for KLC1 expression. Cells were fixed at 24 hpi. ( c ) LD count in mock and infected cells, silenced or not for KLC1. The graph represents the LD count and area measured in pixels of 130 cells for each experimental condition. ( d ) Mock-infected cells transfected with KINTAG, fixed at 24 hpt. ( e ) LD count in KINTAG transfected cells. LD were stained with oil red-O. Bar=10 µm. Representative images of three independent experiments are shown. * P ≤0.005.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Disruption, Infection, Expressing, Transfection, Staining

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Results of Enzyme-Linked Immunosorbent Assays in Different Serum/Plasma Panels

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques:

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Sensitivity and Specificity of Enzyme-Linked Immunosorbent Assays

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques:

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Results of nonstructural protein 1 (NS1) immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) for secondary dengue virus (sDENV) and Zika virus (ZIKV) with previous (wp) DENV infection panels. DENV1-NS1 (A) and ZIKV-NS1 (B) IgG ELISAs in convalescent-phase samples from sDENV, ZIKVwpDENV (Nicaragua [Nic]), probable ZIKVwpDENV (Brazil [Bra]) and negative control (NC) panels. C, Relative optical density (rOD) ratio of ZIKV-NS1 to DENV1-NS1. The sensitivity and specificity are shown based on a cutoff rOD ratio at 0.24. DENV1-NS1 (D) and ZIKV-NS1 (E) IgG ELISAs in post–convalescent phase samples (3 months to 2 years post–symptom onset) from sDENV panels. F, Positive rates of DENV1- and ZIKV-NS1 IgG ELISAs in sDENV panels over time. Dotted lines indicate cutoff rOD values for ELISAs. Data are mean of 2 experiments (each in duplicate). Two-tailed Mann-Whitney test was used to compare the 2 groups.

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques: Virus, Infection, Negative Control, Two Tailed Test, MANN-WHITNEY

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Results of nonstructural protein 1 (NS1) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISAs) in sequential samples from individuals with secondary dengue virus (sDENV) and Zika virus (ZIKV) infection with previous (wp) DENV infections. A, Three cases with sDENV infection, 3–18 months post–symptom onset. B, Four blood donors with sDENV infection, 7 days to 6 months post–index day (PID). C, Five blood donors with ZIKVwpDENV infection, from index day to 3 months PID. Three cases (1, 4, and 7) seroconverted to ZIKV-NS1; 2 cases (15 and 21) had ZIKV-NS1 IgG starting from the index day. Dotted lines indicate cutoff relative optical density (rOD) values for ELISAs. Data are mean of 2 experiments (each in duplicates).

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques: Enzyme-linked Immunosorbent Assay, Virus, Infection

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Proposed algorithm of using 3 serological tests (without neutralization tests) to distinguish different Zika virus (ZIKV) and dengue virus (DENV) infections in dengue- and Zika-endemic regions in the framework of Centers for Disease Control and Prevention guidelines for laboratory diagnosis of ZIKV infection [6, 7]. Only samples tested positive or equivocal by ZIKV or DENV E protein–based immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) are included in the algorithm. The total numbers from each panel and the numbers of positive or negative based on the 3 Nonstructural Protein 1 (NS1) ELISAs are shown in parentheses. Abbreviations: IgG, immunoglobulin G; OD, optical density; pDENV, primary dengue virus infection; pZIKV, primary Zika virus infection; rOD, relative OD; sDENV, secondary dengue virus infection; ZIKVwpDENV, Zika virus infection with previous dengue virus infection.

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques: Neutralization, Virus, Infection