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: bioRxiv

Article Title: Minimally disruptive optical control of protein tyrosine phosphatase 1B

doi: 10.1101/776203

Figure Lengend Snippet: a , Left: An alignment of a competitively inhibited structure of PTP1B (orange, pdb entry 2f71) and an apo structure of PTP1B (yellow, pdb entry 3a5j) highlight an allosteric control system. Closure of the WPD loop (black) over an inhibitor orders the α7 helix; opening of the loop (red) hinders this ordering. Right: An alignment of the LOV2 domain from A. sativa (blue) and an N-terminal segment of the same domain of A. thaliana (white) that is identical between the two proteins (pdb entries 2v0w and 4hhd, respectively). Two terminal α-helices (gray and white) are stable in the dark state, but not the light state. b , Design of a photoswitchable chimera. Light-induced unwinding of the A’α helix of LOV2 destabilizes the α7 helix of PTP1B, causing an allosteric conformational change that inhibits catalysis. We attached the C-terminal α7 helix of PTP1B to the N-terminal A’α helix of LOV2 at crossover points in a primary sequence alignment (1-8). These points are highlighted in blue (PTP1B) and red (LOV2) in a . c , Assays on 4-methylumbelliferyl phosphate (4MUP) show the results of chimera optimization. Construct 7 has the largest dynamic range (DR) of the crossover variants; 7.1 has a higher activity than 7, and 7.1(T406A), termed PTP1B PS , has a larger DR than 7.1. The dashed gray and blue lines denote values for 7.1 and 7.1(T406A), respectively. The plotted data depict the mean, SE, and associated estimates of DR for n = 6 independent experiments. d, Aligned catalytic domains of PTP1B in three structures: photoswitchable (6ntp), apo (3a5j), and competitively inhibited (2f71, α6 and α7 only). e , An analysis of the activity of PTP1B PS on p-nitrophenyl-phosphate (pNPP) indicates that light affects k cat , but not K m ( k cat-dark / k cat-light = 2.50 +/- 0.04). Error bars denote SE for n = 6 independent reactions. f , The DR of PTP1B PS is similar for substrates of different sizes. The plotted data depict the mean, SE, and associated estimates of DR for n ≥ 3 independent reactions. g , Structures of pNPP, 4MUP, and a peptide (PEP) derived from epidermal growth factor receptor (EGFR). Source data are provided as a Source Data file.

Article Snippet: Plasmids harboring important genes used in this study are available from Addgene: LOV2 (pTriEx-PA-Rac1, #22024,) full-length PTP1B (pGEX-2T-PTP1B, #8602), and biosensor (Kras-Src FRET biosensor, #78302).

Techniques: Sequencing, Construct, Activity Assay, Derivative Assay

Journal: bioRxiv

Article Title: Minimally disruptive optical control of protein tyrosine phosphatase 1B

doi: 10.1101/776203

Figure Lengend Snippet: a , Mutations that either prevent adduct formation in LOV2 (C450M), destabilize the A’α and Jα helices (I532E, I539E, and ΔJα), or disrupt the allosteric network of PTP1B (Y152A/Y153A) reduce the photosensitivity of 7.1 and, with the exception of I532E and C450M, lower its specific activity. The plotted data depict the mean, SE, and associated estimates of DR for n = 6 independent experiments. b , Exposure of PTP1B PS to 455 nm light reduces its α-helical content (i.e., the mean residue ellipticity [MRE] at 222 nm). c , An analysis of different chimeras indicates that light-induced changes in α-helical content (i.e., δ 222 = [CD 222-dark -CD 222-light ]/CD 222-dark , or the fractional change in MRE at 222 nm) are necessary, but not sufficient for light-sensitive catalytic activity (i.e., high DR). Mutations correspond to variants of 7.1. Chimeras with large values of δ 222 appear in blue; the dashed line indicates δ 222 for equimolar amounts of free PTP1B and LOV2. Error bars denote SE for n = 6 independent reactions. d-e , Thermal recovery of ( d ) α-helical content (i.e., the change in MRE at 222 nm normalized by the full change over 250 seconds) and ( e ) tryptophan fluorescence (i.e., the change in fluorescence normalized by the full change over 250 seconds) of PTP1B PS . f , A crystal structure of PTP1B (pdb entry 2f71) shows the locations of six tryptophan residues (blue) and the WPD loop (yellow). g , Kinetic constants for thermal recovery are larger for α-helical content than for tryptophan fluorescence (the latter of which is not affected by W491). The discrepancy between these constants is smallest for PTP1B PS (i.e., 7.1(T406A)). The plotted data depict the mean, associated data points, and SE for n = 6 independent reactions. Source data are provided as a Source Data file.

Article Snippet: Plasmids harboring important genes used in this study are available from Addgene: LOV2 (pTriEx-PA-Rac1, #22024,) full-length PTP1B (pGEX-2T-PTP1B, #8602), and biosensor (Kras-Src FRET biosensor, #78302).

Techniques: Activity Assay, Fluorescence

Journal: bioRxiv

Article Title: Minimally disruptive optical control of protein tyrosine phosphatase 1B

doi: 10.1101/776203

Figure Lengend Snippet: a , We assembled PTP1B PS* and PTP1B PS** by attaching residues 299-405 (the disordered proline-rich region) and 299-435 (the proline-rich region and ER anchor), respectively, of full-length PTP1B to the C-terminus of PTP1B PS. Colors correspond to the catalytic domain of PTP1B (orange), the LOV2 domain (blue), the proline-rich region of PTP1B (black), and the ER anchor of PTP1B (gray). b , PTP1B PS * is photoswitchable but exhibits a reduced DR, relative to PTP1B PS ; mutations that stabilize the Jα helix do not improve DR. The plotted data depict the mean, SE, and associated estimates of DR for n = 6 independent experiments. c, Saturation curves show the activity of PTP1B PS* on pNPP ( k cat-dark / k cat-light = 2.03 +/- 0.04). Error bars denote SE for n = 3 independent reactions. d , Images of COS-7 cells expressing GFP-tagged variants of PTP1B. PTP1B 435 and PTP1B PS** exhibit indistinguishable localization patterns (scale bars appear as a white line over a small black rectangle in the lower right corner of each image;10 μm). Source data are provided as a Source Data file.

Article Snippet: Plasmids harboring important genes used in this study are available from Addgene: LOV2 (pTriEx-PA-Rac1, #22024,) full-length PTP1B (pGEX-2T-PTP1B, #8602), and biosensor (Kras-Src FRET biosensor, #78302).

Techniques: Activity Assay, Expressing

Journal: bioRxiv

Article Title: Minimally disruptive optical control of protein tyrosine phosphatase 1B

doi: 10.1101/776203

Figure Lengend Snippet: a, A biosensor for PTP1B activity. Src-mediated phosphorylation of the substrate domain causes it to bind SH2, triggering a conformational change that decreases FRET; dephosphorylation by PTP1B increases FRET. b, Src increases the donor/acceptor emission ratio in vitro (normalized by the buffer-only condition); EDTA or PTP1B prevent this increase. Error bars denote propagated SE for measurements of n = 3 independent experiments (measurements are normalized to a to a buffer-only condition). c, The percent change in donor/acceptor emission ratio over 1 min within 5-μm circular regions located in the cytosol and nucleus of COS-7 cells activated with 457 nm light. Each condition includes the interquartile average, associated data points, and SE for n = 11 biological replicates. The p-values correspond to a two-tailed Student’s t test. d , An image of localized illumination (405 nm) of a COS-7 cell expressing both PTP1B PS and biosensor. Circles delineate irradiated (red) and secondary (blue) regions. e , Time courses of FRET in irradiated and secondary regions. Shading highlights 5-s periods before (gray), during (blue), and after (gray) illumination. f , A depiction of a HEK293T/17 cell expressing PTP1B PS** . Insulin stimulates phosphorylation of the membrane-bound insulin receptor (IR); PTP1B dephosphorylates it. g , ELISA-based measurements of IR phosphorylation in (i) wild-type HEK293T/17 cells and (ii) HEK293T/17 cells stably expressing PTP1B PS** or PTP1B PS** (C450M). Insulin-mediated simulation of IR, BBR-mediated inhibition of PTP1B, and photoinactivation of PTP1B all increase IR phosphorylation. The dark state of PTP1B PS** and the dark and light states of PTP1B PS** (C450M), by contrast, leave IR phosphorylation unaltered from its levels in the wild-type strain (DMSO). The plotted data depict the mean, propagated SE, and associated data points for measurements of n = 3 biological replicates (relative to a buffer-only condition). Source data are provided as a Source Data file.

Article Snippet: Plasmids harboring important genes used in this study are available from Addgene: LOV2 (pTriEx-PA-Rac1, #22024,) full-length PTP1B (pGEX-2T-PTP1B, #8602), and biosensor (Kras-Src FRET biosensor, #78302).

Techniques: Activity Assay, De-Phosphorylation Assay, In Vitro, Two Tailed Test, Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Stable Transfection, Inhibition

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