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: Cell Death & Disease

Article Title: Cryptotanshinone inhibits human glioma cell proliferation in vitro and in vivo through SHP-2-dependent inhibition of STAT3 activation

doi: 10.1038/cddis.2017.174

Figure Lengend Snippet: Inhibitory effect of CTS on p-STAT3 is mediated by SHP-2. ( a ) U251 cells were treated with pervanadate at indicated concentration for 1h, and with additional 10 μ M CTS for 2 h. Proteins were analyzed by western blotting with STAT3, p-STAT3 (Tyr705) and GAPDH antibodies. (PI: phosphoesterase inhibitors, pervanadate). ( b ) U251 cells were treated with 10 μ M CTS for 0, 0.15, 0.5, 1, 2, 4 h. Proteins were analyzed by western blotting with SHP-1, TC-PTP, SHP-2, p-SHP-2 (Tyr542), p-SHP-2 (Tyr580) and GAPDH antibodies. ( c – e ) U251 cells were transfected with specific phosphates siRNA including SHP-1 ( c ), TC-PTP ( d ), SHP-2 ( e ) or negative control (NC) for 48 h, and then treated with CTS with the indicated concentration for 120 min, followed by western blot analysis with the indicated phosphates antibodies. Expression of p-STAT3 (Tyr705) relative to STAT3 were quantified by densitometry. Results are the mean±S.E.M. ( n =3 for each group). ## P <0.01, ### P <0.001 versus control. ( f ) Cell lysates from U251 cells treated with 0, 5, 10 μ M CTS for 2 h were subjected to SHP-2 phosphatase activity assay. Data are expressed as mean±S.E.M., n =3 for each group. * P <0.05; ** P <0.01 versus CTS 0 μ M group. ( g and h ) U251 cells were pretreated with 25 μ M NSC-87877 for 30 min, followed by 10 μ M CTS treatment for 120 min. Total cell lysates were prepared and subjected to western blot analysis with indicated antibodies ( g ) or SHP-2 phosphatase activity assay ( h ). Data are expressed as mean±S.E.M., n =3 for each group. * P <0.05; ** P <0.01, versus CTS 0 μ M group

Article Snippet: Primary antibodies were used as follows: antibodies against SHP-1 and SHP-2 (Epitomics, Burlingame, CA, USA); STAT3, p-STAT3 (Tyr705), p-STAT3 (Ser727), p-p44/42 Erk1/2 (Thr202/Tyr204), cyclin D1, cyclin E1, survivin, p-SHP-2 (Tyr580), p-SHP-2 (Tyr542) and Histon H3 (Cell Signaling Technology); antibody against Ki67 (BD Bioscience); antibodies against TC-PTP, SHP-2, Mcl-1 (Proteintech, Wuhan, Hubei, China); GAPDH (Beyotime).

Techniques: Concentration Assay, Western Blot, Transfection, Negative Control, Expressing, Phosphatase Assay

Journal: Cell Death & Disease

Article Title: Cryptotanshinone inhibits human glioma cell proliferation in vitro and in vivo through SHP-2-dependent inhibition of STAT3 activation

doi: 10.1038/cddis.2017.174

Figure Lengend Snippet: Growth inhibition of intracranial tumor by CTS treatment extended survival of nude mice bearing intracerebral U87 xenografts treated with CTS. ( a ) Growth inhibition of intracranial tumor by CTS treatment. Left: after hematoxylin–eosin staining, tumor size was determined by the areas that were measured at the maximal brain tumor dimensions in the coronal sections. The red circles indicate tumor tissue. Scale bar, 1 mm. Data were calculated by taking the tumor size of control as 100%. Data are expressed as mean±S.E.M., n =5 for each group. * P <0.05; ** P <0.01 versus Model control group. Right: higher magnification in HE staining. Scale bar, 50 μ m. ( b ) After xenografts for 7 days, nude mice were treated with CTS from day 8 to 21 and were observed after discontinuation of therapy. Statistical significance was achieved by Cox–Mantel and Wilcoxon analyses of a Kaplan–Meier survival curve ( n =5). ( c ) Some samples from the harvested tumor tissue on Day 28 were used in detecting tyrosine phosphatase activity of SHP-2. Data are expressed as mean±S.E.M., n =3 for each group. * P <0.05; ** P <0.01, versus Model control group

Article Snippet: Primary antibodies were used as follows: antibodies against SHP-1 and SHP-2 (Epitomics, Burlingame, CA, USA); STAT3, p-STAT3 (Tyr705), p-STAT3 (Ser727), p-p44/42 Erk1/2 (Thr202/Tyr204), cyclin D1, cyclin E1, survivin, p-SHP-2 (Tyr580), p-SHP-2 (Tyr542) and Histon H3 (Cell Signaling Technology); antibody against Ki67 (BD Bioscience); antibodies against TC-PTP, SHP-2, Mcl-1 (Proteintech, Wuhan, Hubei, China); GAPDH (Beyotime).

Techniques: Inhibition, Staining, Activity Assay

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: IRS1/2 are required for insulin-activated IR endocytosis. a HepG2 cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP antibodies. b Quantification of the ratios of PM and IC IR-GFP signals of cells in ( a ) (mean ± SD; *p<0.0001). c Domains and YXXΦ motifs of human IRS1 and mouse IRS2. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. AP2M1- and SHP2-binding regions are indicated. YXXΦ motifs and phosphotyrosine sites of IR for SHP2 binding are shown as blue and red bars, respectively. The MAPK phosphorylation sites are labeled as green letters in the sequences. d 293FT cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP (IR; green), anti-Myc (IRS1; red), and DAPI (blue). (3YA, Y612A/Y632A/Y662A; 3YF, Y612F/Y632F/Y662F; 3SA, S616A/S636A/S666A; 3SD, S616D/S636D/S666D; Y2A, Y1179A/Y1229A). e Quantification of the ratios of PM and IC IR-GFP signals of cells in ( d ) (mean ± SD; *p<0.0001). f 293FT cells were serum starved and treated without or with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP, and IgG IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. g Serum-starved primary hepatocytes were treated with DMSO or 10 µM SHP099 for 2 h and treated with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP were blotted with anti-IRS1 and anti-AP2B1 antibodies.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: IRS1 promotes IR endocytosis and interacts with AP2. a Western blot analysis of cell lysates in . Asterisks indicate non-specific bands. b Domains and YXXΦ motifs of human IRS1. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. IRS1 fragments that can or cannot bind to AP2M1 are presented as red or black lines, respectively. YXXΦ motifs and phosphotyrosine sites for SHP2 binding are presented as blue and red bars, respectively. c Binding of IRS1 WT and mutants to GST or GST-AP2M1. Input and protein bound to beads were blotted with anti-Myc (IRS1) antibodies and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± SD; n=3 independent experiments). d Binding of IRS1 WT and truncation mutants to GST or GST-AP2M1. Input and protein bound to beads were blotted with the indicated antibodies. The relative band intensities are shown below (n=2 independent experiments). e Sequence alignment of a conserved region in IRS1/2. Three YXXΦ motifs are boxed with red dashed lines. The phosphorylation sites of IR and MAPK are indicated as red and blue dots, respectively.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Western Blot, Binding Assay, Staining, Sequencing

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: The SHP2-MAPK pathway promotes insulin-activated IR endocytosis. a HepG2 cells expressing IR-GFP WT were starved, treated with the indicated inhibitors for 2 h, treated without or with 100 nM insulin for 20 min, and stained with anti-GFP (IR; green) and DAPI (blue). b Quantification of the ratios of PM and IC IR-GFP signals of cells in (A) (mean ± SD; *p<0.0001). c Binding of IRS1 peptides to AP2M1 (residues 160-435). Input and proteins bound to IRS1-peptide beads were analyzed by SDS-PAGE and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± SD; n=4 independent experiments). d Isothermal titration calorimetry (ITC) analysis of binding between IRS1 peptides and AP2M1 (residue 160-435), with K d indicated. e The IRS1 peptides were incubated with active SHP2 for the indicated durations, spotted onto membranes, and detected with the anti-pY612-IRS1 antibody. f Quantification of the relative SHP2 activity in ( e ) (mean ± SD; n=4 independent experiments; *p<0.0001). g Model of the regulation of insulin-activated IR endocytosis by a phosphorylation switch on IRS1/2. Insulin-bound IR phosphorylates itself and IRS1/2, and activates the PI3K-AKT and MAPK pathways. SHP2 acts upstream of RAS-RAF and promotes the activation of MAPK pathway. p31 comet binds to the IR-bound MAD2 and blocks IR-AP2 association to prevent premature IR endocytosis. In feedback regulation, activated ERK1/2 phosphorylate S616 and other sites on IRS1. SHP2 binds to the C-terminal phospho-tyrosine site on IRS1 and dephosphorylates pY612 of the doubly phosphorylated IRS1 (pY612/pS616), thus promoting IRS1-AP2M1 association. p31 comet is released from MAD2 by an unknown mechanism, allowing the assembly of an MCC-like complex on IR. MAD2- and IRS1/2-dependent AP2 recruitment and clustering trigger clathrin-mediated IR endocytosis. h Ribbon diagram of the crystal structure of AP2M1 (residues 160-435) bound to pS-IRS1. pS-IRS1 is shown as sticks. i Surface drawing of AP2M1, with pS-IRS1 shown as sticks. j A close-up view of the surface drawing of AP2M1 colored by its electrostatic potential (blue, positive; red, negative; white, neutral). pS-IRS1 is shown as sticks.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Expressing, Staining, Binding Assay, SDS Page, Isothermal Titration Calorimetry, Incubation, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: SHP2 inhibition delays IR endocytosis and improves insulin sensitivity in mice. a , b Glucose tolerance test ( a ) and insulin tolerance test ( b ) of male mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. At 1 day after the last drug administration, experiments were performed. Vehicle, n=12; SHP099, n=10; mean ± SEM. c Body weight of mice administered vehicle or SHP099 at 7 days post administration. Mean ± SD. d HFD-fed mice were administered vehicle or SHP099 for 5 days. The mice were fasted overnight and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 µm. e Quantification of the ratios of plasma membrane (PM) and intracellular compartments (IC) IR signals of the livers in ( d ) (mean ± SD; *p<0.0001). f - h The levels of fasting serum insulin ( f ) and C-peptide ( g ), and the ratio of C-peptide:insulin ( h ) in mice fed normal chow or HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. i HepG2 cells stably expressing IR-GFP were transfected with CEACAM1 siRNAs, serum starved, treated without or with 100 nM insulin form 5 min, and stained with anti-GFP and DAPI. Quantification of the ratios of PM and IC IR-GFP signals of cells was shown (mean ± SD). j Western blot analysis of cell lysates in ( i ). k Model of the regulation of insulin-activated IR endocytosis by CEACAM1, the MAD2–CDC20– BUBR1 module, and the SHP2-IRS1/2 module.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Inhibition, Injection, Staining, Stable Transfection, Expressing, Transfection, Western Blot

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: Depletion of SHP2 by shRNA delays IR endocytosis and improves insulin sensitivity in mice. a The level of SHP2 in liver, skeletal muscle and epididymal WAT from mice fed HFD for 5 weeks. The mice were injected with AAV-control (Ctrl) or SHP2 shRNA. At 17 days after injection, the mice were fasted overnight and injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. WAT and skeletal muscle were collected at 2 min and 3 min after the indicated time points, respectively. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b , c Glucose tolerance test ( b ) and insulin tolerance test ( c ) in mice injected with AAV-Ctrl or AAV-SHP2 shRNA and fed HFD. Experiments were performed at 2 weeks after injection. n=6; mean ± SEM. d Body weight in HFD-fed mice injected with AAV-Ctrl or AAV-SHP2 shRNA. Mean ± SD. e HFD-fed mice were injected with AAV-Ctrl or AAV-SHP2. At 17 days after injection, the mice were fasted overnight and injected with or without 1U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 µm. f Quantification of the ratios of PM and IC IR signals of the livers in ( e ) (mean ± SD; *p<0.0001).

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: shRNA, Injection, Western Blot, Staining

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: Mitotic regulators and SHP2 promote feedback inhibition of IR. a Insulin signaling in the liver from mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 5 days, fasted overnight, and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b Quantification of the blots in ( a ). Mean ± SD; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. c Targeting feedback regulation of IR endocytosis for diabetes treatment. Left panel depicts the feedback regulation of IR endocytosis by ERK1/2 and SHP2 during unperturbed insulin signaling. Right panel illustrates the mechanism by which SHP2 inhibitor (SHP099) or shRNA blocks growth-promoting IR signaling and IR endocytosis, and prolongs insulin signaling through the PI3K-AKT pathway, which controls metabolism.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Inhibition, Injection, Western Blot, shRNA

Journal: Histochemistry and Cell Biology

Article Title: Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice

doi: 10.1007/s00418-020-01905-6

Figure Lengend Snippet: Distribution of TBC proteins in the organ of Corti at P1 and P7. a – e age P1; a TBCA expression is found in the inner area of Kölliker’s organ, the inner hair cell and a tectal cell. b TBCB is expressed in the cells of the Kölliker’s organ. c TBCC labelling is detected in the basal half of the two pillar cells. d Similar TBCD is expressed in the pillar cells. e TBCE staining is found in the basal cells of Kölliker’s organ and the basal half of the inner phalangeal cell. Diffuse staining is visible around the nucleus and below the apical surface of the inner hair cells. The three outer hair cells are also marked. f – j age P7: f TBCA is detected in the inner and outer pillar cells, phalangeal extensions of the three Deiters’ cells, tectal cells and Hensen’s cells. g Similar to TBCA, TBCB is detected in the inner and outer pillar cells, the three Deiters’ cells, the tectal cells and Hensen’s cells. h The expression of TBCC is found in the supporting cells (inner and outer pillar cells, the phalangeal extensions of the three Deiters’ cells, the tectal cells and Hensen’s cells). i TBCD staining is found in the basal parts of the inner pillar cell. j In addition to the inner pillar cells, TBCE is also detected in the and the basal half of the inner phalangeal cell phalangeal extensions of Deiters’ cells. ( TM tectorial membrane with unspecific staining) (Scale bar = 25 µm)

Article Snippet: For immunofluorescence analysis, the tissue was blocked in 10% BSA in 0.1% Triton X-100 for 1 h. Primary antibodies were incubated in 1% BSA in 0.1% Triton X-100 overnight at 4 °C at the following concentrations: mouse monoclonal against β-tubulin (1:500, Sigma- Aldrich), rabbit polyclonal against TBCA (1:50), TBCB (1:100), TBCC (1:200), TBCD (1:100) and TBCE (1:100, all Proteintech).

Techniques: Expressing, Staining, Membrane

Journal: Histochemistry and Cell Biology

Article Title: Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice

doi: 10.1007/s00418-020-01905-6

Figure Lengend Snippet: Distribution of TBC proteins in the organ of Corti at P14. a TBCA labelling is found in both the tectal and Hensen’s cells. β-Tubulin stains the phalangeal extension of the Deiters’ cells. b TBCB expression is detected in basal cell half of the Deiters’ cells, the phalangeal processes are stained by β-tubulin. c The expression of TBCC is found in the apical part of the inner pillar cell, β-tubulin in the Deiters’ cells. d Staining of TBCD is visible in the cell body of outer hair cells. Phalloidin stains the cuticular plate and stereocilia of the outer hair cells and the inner pillar cell. e TBCE is detected in the outer hair cells, whereas Phalloidion marks the cuticular plate and the stereocilia of the outer hair cells and a bundle-like structure extending from the apex to the base of inner pillar cell. (Scale bar = 25 µm)

Article Snippet: For immunofluorescence analysis, the tissue was blocked in 10% BSA in 0.1% Triton X-100 for 1 h. Primary antibodies were incubated in 1% BSA in 0.1% Triton X-100 overnight at 4 °C at the following concentrations: mouse monoclonal against β-tubulin (1:500, Sigma- Aldrich), rabbit polyclonal against TBCA (1:50), TBCB (1:100), TBCC (1:200), TBCD (1:100) and TBCE (1:100, all Proteintech).

Techniques: Expressing, Staining

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Comparison of non-structural protein 1 sequences among closely related flaviviruses. ( A ): Ribbon model highlighting regions of the NS1 protein containing segments exposed at the outer surface to the host environment. ( B ): Sequence comparison showing regions with high sequence disparity. Amino acids depicted in red differ from the corresponding ZIKV NS1 amino acids. A represents positions with two sequences with amino acids identical to ZIKV NS1. The boxes highlight highly conserved sequences, amino acids 117–119 and 227–229, that were mutated to alanine in immunodominant regions 2 and 3.

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Comparison, Sequencing

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Western blot of NS1 mutants. ( A ): The blot was probed with anti-His6 antibody targeted toward the protein N-terminus. ( B ): The blot was probed with anti-ZIKV NS1 monoclonal antibody targeted toward the C-terminus. Uncropped gels are displayed in .

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Western Blot

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Ratios of mutant vs. wild-type binding of NS1 by patient samples. The blue line at 1 indicates equal binding. Numbers over 1 indicate increased binding to the named mutant, while under 1 indicate increased wild-type binding. ( A ): Ratios of mutant/wild-type binding from IgG in samples show preferential binding to 117–119 mutant NS1. ( B ): IgM shows preferential binding of mutant NS1 proteins in samples from the Dominican Republic. Colombia = Colombia samples from suspected ZIKV infection. Dom Rep = Dominican Republic samples from suspected ZIKV infection. 117–119 = ZIKV NS1 W117A, G118A, K119A. 227–229 = ZIKV NS1 H227A, T228A, L229A Comparisons were made using Kruskal–Wallis ANOVA. Asterisks represent significant comparisons (**** p < 0.0001; ** p < 0.01). The full dataset is presented in .

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Mutagenesis, Binding Assay, Infection

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Serum from goats immunized with ZIKV mutants show differential binding to NS1 proteins. Antibody-capture ELISA was performed on a mixture of serum from two goats immunized with ZIKV NS1 protein. Binding of WT ZIKV NS1 showed lower binding to WT ( A ), both mutants ( B , C ), as well as DENV2 WT NS1 ( D ). Data represent samples run in duplicate. Error bars (standard error of the mean) were smaller than the symbols as drawn. The experiment was repeated a total of three times. The legend in panel ( D ) also applies to panels ( A – C ).

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Protein Binding

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Process for purifying and cross-adsorbing ZIKV NS1 antibodies. ZIKV NS1 antibodies were affinity purified using a gravity flow column. ( A ): The bound and eluted fraction was put through a column with DENV NS1 twice. The unbound and eluted (non-DENV reactive) fraction is the cross-adsorbed, ZIKV-specific fraction. ( B ): Antibodies flowing through the column initially bind ZIKV at low signal strength. Following ZIKV NS1 column elution, strength of binding of the solution goes up, but DENV NS1 reactivity is present. Upon cross-adsorption against DENV NS1, specificity of the pAb solution goes up, as well as its binding avidity to ZIKV NS1.

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Affinity Purification, Binding Assay, Adsorption

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: ZIKV NS1 antigen-capture ELISA limit of detection and dynamic range. ( A ): ZIKV rNS1 shows binding significantly higher than BSA down to 7.8 ng/mL. The limit of detection of the assay is between 7.8 and 1.95 ng/mL NS1. ( B ): Linear regression of NS1 detection via ELISA demonstrates assay can quantify protein across a range of values. Comparisons were made using two-way ANOVA with a Holm–Sidak multiple comparisons test. Asterisks represent significant comparisons (**** p < 0.0001; * p < 0.05). Error bars represent the standard error of the mean.

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Production of flavivirus NS1. Coomassie-stained SDS-PAGE images of induced (I) and uninduced (U) cultures from NS1 proteins of yellow fever virus ( A ), West Nile virus ( A , B ) and St. Louis Encephalitis virus ( B ). Bands indicate proteins similar to DENV and ZIKV NS1 WT production. Results from three different colonies [ , , ] for each flavivirus NS1 are indicated. Uncropped gels are displayed in .

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Staining, SDS Page, Virus

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: The ZIKV NS1 antigen-capture assay demonstrates low cross-reactivity to related viruses. Recombinant NS1 antigens were produced and assayed. A: Even at low levels of NS1 detection, the assay is specific to ZIKV NS1. Comparisons made using two-way ANOVA with a Holm–Sidak multiple comparisons test. Asterisks represent significant comparisons between ZIKV NS1 and all other NS1 proteins (**** p < 0.0001; * p < 0.05). Error bars represent the standard error of the mean.

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Recombinant, Produced

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: ZIKV NS1 antigen-capture ELISA results from patients infected during the 2015–2016 outbreaks in Colombia and the Dominican Republic 1 .

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Infection