recombinant denv2 ns1 protein  (R&D Systems)

Journal: PLOS Pathogens

Article Title: The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection

doi: 10.1371/journal.ppat.1012167

Figure Lengend Snippet: (A) BMDMs were primed with PAM 3 CSK 4 (1μg/mL) for 17 h and then treated with DENV2 NS1 at indicated concentrations, treated with 5μM nigericin, or left untreated (PAM only). IL-1β levels in the supernatant after 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. Statistical significance was determined by one-way ANOVA with Dunn’s multiple compa rison correction. (B) Representative Western blots of BMDM cell lysates after priming with PAM 3 CSK 4 (1μg/mL) for 17h and treatment with 10ug/mL DENV2 NS1 (DENV NS1) or PAM 3 CSK 4 treatment for 24h without NS1 treatment (PAM only). (C) WT and Casp1/11 -/- BMDMs were primed with PAM 3 CSK 4 (1μg/mL) for 17h and then treated with DENV2 NS1 at the indicated concentrations, nigericin (5μM), or medium (PAM only). IL-1β levels in the supernatant after 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. Statistical significance was determined using two-way ANOVA with Holm-Sidak’s multiple comparisons test. (D) BMDMs were primed with PAM 3 CSK 4 (1μg/mL) for 17h and then pre-treated with Ac-YVAD-cmk at the indicated concentrations before addition of DENV2 NS1 (10μg/mL), nigericin (5μM), or medium (Inhibitor only). IL-1β levels in supernatant 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. (E) WT or Casp-1 -/- THP-1 human monocytes were differentiated into macrophages in 10ng/mL PMA and primed with medium or LPS for 4h. Primed macrophages were treated with DENV NS1 (10μg/mL) or left untreated (LPS only). Eighteen hours later, supernatants were collected. Cells were stimulated with 5μM nigericin for 2h as a positive control. Bioactive IL-1 in supernatants was measured using HEK-Blue IL-1R reporter cells. Statistical significance was determined using two-way ANOVA with Holm-Sidak’s multiple comparisons test (F) Representative Western blots of THP-1 macrophage cell lysates after priming with PAM 3 CSK 4 (100ng/mL) for 17h and treatment with DENV2 NS1 at indicated concentrations (μg/mL), treatment with 5μM nigericin, or no treatment for 24h. The data are shown as the mean ± standard deviation (SD) of 3 independent experiments (A, C-D) or 4–6 independent experiments (E) or a representative image taken from 2 biological replicates (B, F). *p<0.05, **p<0.01, *** p< 0.001, ****p<0.0001.

Article Snippet: Recombinant DENV2 NS1 (Thailand/16681/84) was produced in mammalian HEK293 cells or purchased from The Native Antigen Company (Oxford, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Positive Control, Standard Deviation

Journal: PLOS Pathogens

Article Title: The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection

doi: 10.1371/journal.ppat.1012167

Figure Lengend Snippet: (A) WT and Nlrp3 -/- BMDMs were primed with PAM 3 CSK 4 (1μg/mL) for 17h and then treated with DENV2 NS1 at indicated concentrations, nigericin (5μM), or medium (PAM only). IL-1β levels in supernatant 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. Statistical significance was determined using two-way ANOVA with Holm-Sidak’s multiple comparisons test. (B) Representative Western blots of cell lysates from WT and Nlrp3 -/- BMDMs after priming with PAM 3 CSK 4 (1μg/mL) for 17h and treatment with DENV2 NS1 (10 or 5 μg/mL), treatment with nigericin (5μM), or no treatment for 24h. (C) BMDMs were primed with PAM 3 CSK 4 (1μg/mL) for 17h and then pre-treated with MCC950 at the indicated concentrations before addition of DENV2 NS1 (10μg/mL), nigericin (5μM), or medium (Inhibitor only). IL-1β levels in the supernatant after 2h (Nigericin) or 24h (NS1 and PAM only) were measured by ELISA. (D) Representative Western blots of cell lysates from BMDMs nucleofected with Cas9-gRNA ribonuclear protein complexes to knock out the indicated genes. Two gRNAs per gene were used per nucleofection. NTG = non-targeting guide. ( E) Knockout BMDMs from (D) were primed with PAM 3 CSK 4 (1μg/mL) for 17h and treated with DENV2 NS1 (10μg/mL) or left untreated for 48h. Statistical significance was determined using two-way ANOVA followed by with Holm-Sidak’s multiple comparisons test. The data are shown as the mean ± SD of 3 biological replicates (A,C), a representative image taken from 2 biological replicates (B,D), or data pooled from 8 independent experiments with at least 3 biological replicates per guide (E). *p<0.05, **p<0.01, *** p< 0.001, ****p<0.0001, ns (not significant), p> 0.05.

Article Snippet: Recombinant DENV2 NS1 (Thailand/16681/84) was produced in mammalian HEK293 cells or purchased from The Native Antigen Company (Oxford, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Knock-Out

Journal: PLOS Pathogens

Article Title: The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection

doi: 10.1371/journal.ppat.1012167

Figure Lengend Snippet: (A) BMDMs were primed with PAM 3 CSK 4 (1μg/mL) for 17h and then treated with DENV2 NS1 (10μg/mL), nigericin (5μM), or medium (PAM only). At the indicated timepoints, supernatants were assessed for lactase dehydrogenase (LDH) levels as a proxy for cell death. LDH levels were calculated as a percentage of maximum LDH release. (left panel) Cells were also stained using a LIVE/DEAD Fixable Far Red stain and analyzed by flow cytometry. (right panel) Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparisons test. (B) BMDMs were primed with PAM 3 CSK 4 (1μg/mL) for 17h and then treated with DENV2 NS1 (10μg/mL), LPS (5μg/mL) or no treatment (Untreated). After 30 min, cells were stained for surface CD14 expression and analyzed by flow cytometry. Data are normalized as a percentage of the median fluorescence intensity of the treatment groups divided by the untreated control. Statistical significance was determined using one-way ANOVA with Holm-Sidak’s multiple comparisons test. (C) Representative Western blots of cell lysates from BMDMs nucleofected with either NTG or CD14 Cas9-gRNA ribonuclear protein complexes. (D) BMDMs from (C) were primed with PAM 3 CSK 4 (1μg/mL) for 17h and treated with DENV2 NS1 (10μg/mL) or no treatment for 48h. IL-1β levels in supernatant were measured by ELISA (E). Lysates from cells 24 hours post-treatment were evaluated by Western Blot. Statistical significance was determined using two-way ANOVA with Holm-Sidak’s multiple comparisons test. The data are shown as the mean ± SD of 3 biological replicates (A,B,D), or a representative image taken from 2 biological replicates (C,E). *p<0.05, **p<0.01, *** p< 0.001.

Article Snippet: Recombinant DENV2 NS1 (Thailand/16681/84) was produced in mammalian HEK293 cells or purchased from The Native Antigen Company (Oxford, UK).

Techniques: Staining, Flow Cytometry, Expressing, Fluorescence, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection

doi: 10.1371/journal.ppat.1012167

Figure Lengend Snippet: (A-B) Survival curves (A) and weight loss over time (B) of Casp1/11 +/+ Ifnar -/- (Caspase 1/11 WT), Casp1/11 +/- Ifnar -/- (Caspase 1/11 Het), or Casp1/11 -/- Ifnar -/- (Caspase 1/11 KO) littermates infected intravenously with 3 x 10 5 PFU of DENV2 D220. Survival was monitored over 14 days. Weight loss was monitored over 9 days. Numbers in parentheses indicate the numbers of mice in each group. Statistical significance was determined by Mantel–Cox log-rank test (A) or two-way ANOVA with Holm-Sidak’s multiple comparisons test (B) . (C-D) Survival curves of Nlrp3 +/+ Ifnar -/- (NLRP3 WT), Nlrp3 +/- Ifnar -/- (NLRP3 Het), or Nlrp3 -/- Ifnar -/- (NLRP3 KO) littermates infected intravenously with a 5 x 10 5 PFU ( High Dose) (C) or 7.5 x 10 4 PFU ( Low Dose) (D) of DENV2 D220 and monitored over 10 days. Numbers in parentheses indicate the numbers of mice in each group. **p<0.01, *** p< 0.001, ns (not significant), p> 0.05.

Article Snippet: Recombinant DENV2 NS1 (Thailand/16681/84) was produced in mammalian HEK293 cells or purchased from The Native Antigen Company (Oxford, UK).

Techniques: Infection

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) BHK-21 cells were infected with DENV-2, fixed at the indicated times post-infection, and processed for confocal microscopy. NS1 (green) and nuclei (DAPI, blue). B) Quantification by two different bioinformatics software, ICY © (upper panel and ImageJ (lower panel), of the amount of NS1 present inside the cell nuclei. Results are expressed as a percentage, taking as 100% the total pool of NS1 present inside the cell (right panels). **/*** p=0.005. There are no significant differences between the two methods

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Infection, Confocal Microscopy, Software

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: BHK-21 cells were infected with DENV-2, fixed at 24 hpi, and examined using confocal microscopy. A) C olocalization of NS1 with cell structures of interest. B) Labelling of different cell proteins for fraction quality control. Sequential removal of different cell parts was achieved throughout the process based on buffer compositions. DIC, differential interference contrast. Results from one of two independent experiments are shown.

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Infection, Confocal Microscopy, Control

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: BHK-21 cells were infected with DENV2 or mock - infected, harvested at 24 hpi, and subjected to subcellular fractionation. (A) Localization of DENV2 NS1 by western blot after fully denaturing SDS-PAGE electrophoresis, or (B) after non-denaturing blue native PAGE electrophoresis. (C) Western blot for quality control of each fraction using appropriate protein markers. Results from one of two independent experiments are shown.

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Infection, Fractionation, Western Blot, SDS Page, Electrophoresis, Blue Native PAGE, Control

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: (A) BHK-21 cells were infected with DENV2 and processed at 24 hpi. Colocalization was analyzed by confocal microscopy, detailing the spatial interaction between NS1 (green) and the nuclear lamina (red). Signal overlap was quantified by Pearson’s (0.162) and Manders (0.324) correlation coefficients. (B) Co-immunoprecipitation assay from BHK-21 cells transfected with a plasmid expressing DENV2 NS1. Cells were collected at 24 hpt and precipitation was performed using NS1 as a bait. Results from one of two independent experiments are shown.

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Infection, Confocal Microscopy, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) Bipartite NLS sequence predicted in DENV-2 NS1 by NLS mapper (score of 5.9, with a total of 1.0 cytoplasmic location and 10.0 nuclear location). B) Sequence alignment of the predicted bipartite NLS across DENV serotypes 1 to 4 and Zika virus. C) Structural models of dimeric (left) and hexameric (right) DENV-2 NS1 generated by Alphafold 3.0 showing the exposed nature of the predicted NLS (highlighted in red and N-glycosylation site (N207) in blue).

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Sequencing, Virus, Generated, Glycoproteomics

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) Mutations introduced in part 1 (Mut1), part 2 (Mut2) or both parts (Mut3) of the bipartite NLS (underlined in red). Residue changes and positions are indicated. B) Evaluation of the amount of NS1 present in the cell nuclei after transfection with the different mutant constructions. Left panels, plasmid expressing GFP with 4 NLS (4-GFP NLS) used as a positive control. Right panels, wild type (pNS1) and the 3 different mutant constructions. Ivermectin treatment of 4-GFP NSL and pNS1-transfected cells was included as an additional positive control. Confluent monolayers of BHK-21 cells were processed for confocal microscopy 24 hpt. Cell nuclei were stained with DAPI (blue), and NS1 was visualized with Alexa488 (green). C) Quantification of the amount of NS1 present inside the cell nuclei, expressed as a percentage of the total signal. For each construction, 30 cells were analyzed. *p<0.05

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Residue, Transfection, Mutagenesis, Plasmid Preparation, Expressing, Positive Control, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) Monolayers of BHK-21 were simultaneously transfected with in vitro transcribed RNA from DENV2-mut-mCherry, together with plasmids that express wild-type NS1 (p wt-NS1) or NS1 mutated in both parts of the NSL (p Mut3-NS1), and 6 days after, cells were fixed and processed for confocal microscopy. Controls include non-transfected cells (N/T), cells transfected with a plasmid expressing wt-NS1 wild type alone (p wt-NS1), and RNA derived from either infectious clone alone (DENV2-wt-mCherry and DENV2-mut-mCherry). B) Cell supernatants collected from the trans complemented cells after 6 days were tested for infectivity in monolayers of BHK-21 cells. Cells were fixed and processed for confocal microscopy 7 days after infection. Cell nuclei were stained with DAPI (blue), and DENV NS1 was visualized using an anti-NS1 Mab as primary antibody and an Alexa 488 conjugated secondary antibody (green). Scale bar: 50uM. Results from one of two independent experiments are shown.

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Transfection, In Vitro, Confocal Microscopy, Plasmid Preparation, Expressing, Derivative Assay, Infection, Staining

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) Volcano plot showing differential gene expression in BHK-21 cells expressing the NS1-M3 mutant relative to wild-type NS1-Wt. The x-axis represents the fold change (log₂), and the y-axis indicates statistical significance (-log₁₀ crude P-value). Orange dots denote significantly upregulated genes in NS1-M3 compared to NS1-Wt, while blue dots represent downregulated genes (log₂ FC ≥ 1, P < 0.05). The extensive distribution of differentially expressed genes highlights the distinct biological impact of the M3 mutation compared to the native protein. B) Bidirectional hierarchical clustering heatmap of 1,755 genes with significant differential expression (times change > 2 and cutoff P-value). Rows represent individual genes and columns represent experimental conditions. Expression levels are presented as Z-scores of log2 normalized values, ranging from low expression (blue) to high expression (yellow). The upper dendrogram reveals a distinct clustering of the NS1-M3 mutant, separate from the NS1-Wt and control groups. (Right)

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Gene Expression, Expressing, Mutagenesis, Quantitative Proteomics, Control

Journal: Nature Communications

Article Title: SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

doi: 10.1038/s41467-022-34910-5

Figure Lengend Snippet: A Schematic depicting S-triggered barrier dysfunction measured by a trans-endothelial/epithelial electrical resistance assay (TEER; left) and an endo/epithelial glycocalyx layer (EGL) assay (right). B Time course of TEER assay measuring the barrier function of HPMEC monolayers over time with the indicated treatments, including DENV2 NS1 (5 µg/mL), VEGF (50 ng/mL), and SARS-CoV-2 S (10 µg/mL). Data are from n = 3 biological replicates. C A TEER assay measuring the barrier of monolayers of HPMEC and HPMEC/ACE2 at 24 hours after the indicated treatments. VEGF positive control (50 ng/mL). Data are from n = 4 biological replicates. D Same as C but treated with the indicated VSV-pseudotyped particles at the indicated dilutions. VSV-bald and VSV-G are diluted 1:30. Data are from n = 4 biological replicates. E Same as C but treated with the indicated concentrations of SARS-CoV-2 RBD. Data are from n = 3 biological replicates. F Same as C but measuring the barrier of Calu-3 cell monolayers. Data are from n = 3 biological replicates. G A TEER inhibition assay measuring the capacity of a cocktail of anti-S antibodies to inhibit S-mediated endothelial hyperpermeability. S (10 µg/mL) and the antibody cocktail (15 µg/mL of each antibody; 1A9 [Genetex] and CR3022 [Absolute Antibody]) were added simultaneously to the upper chamber of Transwell inserts to a monolayer of HPMEC or HPMEC/ACE2, and TEER was measured 24 hours post-treatment (hpt). Data are from n = 3 biological replicates. In all panels, the dotted line is the normalized TEER value of the untreated control condition. All data are plotted as mean + /− SEM. For all panels, values are compared to untreated controls by One-Way ANOVA with Tukey’s Multiple comparisons test except for ( B ), which was analyzed by two-sided unpaired t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, and n.s. p > 0.05. Source data are provided as a Source Data file.

Article Snippet: Recombinant DENV serotype 2 NS1 was purchased from the Native Antigen Company and characterized previously (Dengue virus serotype 2 NS1 [accession # P29990.1, Thailand/16681/84]) .

Techniques: Positive Control, Inhibition

Journal: Nature Communications

Article Title: SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

doi: 10.1038/s41467-022-34910-5

Figure Lengend Snippet: A A representative mouse back from a dermal leak experiment. The dorsal dermises of mice were injected intradermally with the treatments and doses indicated. Mice then received a dextran-680 tracer molecule intravenously. Mouse dermises were collected 2 h post-treatment, and quantification of local dermal leak was assessed by a fluorescent scanner. B Quantification of A from mice treated with PBS ( n = 27), DENV2 NS1 (15 µg; n = 5), S (10 µg; n = 25), and S (25 µg; n = 5). C Representative lung images from a SARS-CoV-2 S systemic vascular leak assay. Mice were administered 50 µg of SARS-CoV-2 S or ovalbumin intranasally as indicated, and 22 hpt were administered a dextran-680 tracer intravenously as in A . Organs of mice were collected 2 hours post dextran-680 administration (24 hours post-S treatment), and accumulation of dextran-680 was measured with a fluorescent scanner. D Quantification of C from n = 6 mice. E Same as C except representative images of spleens are shown. F Quantification of E from n = 6 mice. G Same as C except representative images of small intestine are shown. H Quantification of G from n = 5 mice. I , J Hematoxylin and eosin (H&E) staining was performed on lung sections from K18-hACE2 mice 7 days post-infection with 100 TCID 50 units of SARS-CoV-2 WA/1 isolate. Displayed are representative images of lungs from n = 3 mice in mock-infected conditions ( I ) and from n = 4 mice infected with SARS-CoV-2 ( J ); left panels with scale bars at 2 mm and right panels consisting of zoomed-in insets with scale bars at 100 µm. Arrows point to dispersed red blood cells. K Representative lung images from C57BL/6 mice infected with the indicated dose of SARS-CoV-2 mouse-adapted strain (MA-10) for 7 days. Mice were administered a dextran-680 tracer intravenously as in A . Lungs were collected 2 hours after dextran-680 administration and fixed overnight in formalin, and the fluorescence accumulation was measured with a fluorescent scanner. L Quantification of K from n = 5 mice, except for the 2 × 10 4 PFU condition, which was from n = 4 mice. MFI is mean fluorescence intensity. All data are plotted as mean + /− SEM with * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant DENV serotype 2 NS1 was purchased from the Native Antigen Company and characterized previously (Dengue virus serotype 2 NS1 [accession # P29990.1, Thailand/16681/84]) .

Techniques: Injection, Staining, Infection, Fluorescence