Journal: Scientific Reports

Article Title: Regulation of Angiopoietin Signalling by Soluble Tie2 Ectodomain and Engineered Ligand Trap

doi: 10.1038/s41598-017-03981-6

Figure Lengend Snippet: Mathematical model for kinetics of Ang1 binding to Tie2. ( a ) Schematic diagram of Ang1 interactions represented in Simbiology Toolbox, Matlab. A two-compartment model was constructed with Ang1 binding to sTie2 and the initial recruitment of Ang1 to cellular Tie2 occurring in the extracellular compartment. Recruitment of additional cellular Tie2 receptors occurs in the membrane compartment. Ang1 is represented by A, sTie2 by S, and cellular Tie2 by M. The suffix following A indicates the available receptor binding sites and the suffix following M and S the number of receptors bound to each tetrameric ligand. ( b ) Representation of the space in which Ang1 bound to a cell surface receptor can search for further membrane receptors. The distance above the membrane that the ligand can search is designated by r and represents the distance between binding sites on maximally extended Ang1. ( c ) Schematic representation of Ang1 showing super-clustering (SCD), coiled-coil (CCD) and fibrinogen related domain (FReD) of a monomer together with amino acid numbers at domain junctions, secretory leader domain (residues 1–19) not shown. Below: Structure of Ang1 fibrinogen related domain that contains the C-terminal receptor binding domain is shown together with length indicator. Structure visualized with PYMol. ( d ) Estimation of Tie2 number per cell. Upper blot shows an example of an anti-Tie2 immunoblot used for quantification. Numbers 1–4 designate four different cell lysates and tracks to the right of these are known amounts of recombinant Tie2. Below the blot is an example of a standard curve derived from the blot and used to calculate total Tie2 content of HUVECs. Beneath the standard curve is an example of a blot to determine percentage distribution of cellular Tie2 exposed on the cell surface. Surface Tie2 was labelled with cell impermeant NHS-LC-biotin and biotinylated surface receptor was recovered with streptavidin-agarose. Aliquots of total (T), intracellular (I) and cell surface (S) fractions were resolved by SDS-PAGE and probed for Tie2 or the intracellular protein tubulin, by immunoblotting, as indicated. Images of blots have been cropped for clarity.

Article Snippet: Recombinant human Angpt1, antibodies recognizing Tie2 extracellular domain and anti-phosphoY 992 -Tie2 antibody were obtained from R&D Systems.

Techniques: Binding Assay, Construct, Membrane, Cell Surface Receptor Assay, Western Blot, Recombinant, Derivative Assay, SDS Page

Journal: Scientific Reports

Article Title: Regulation of Angiopoietin Signalling by Soluble Tie2 Ectodomain and Engineered Ligand Trap

doi: 10.1038/s41598-017-03981-6

Figure Lengend Snippet: Numerical simulations of Ang1 binding to cellular Tie2. The mathematical model described in Supplementary Information was utilized to examine the time course of formation of cell surface complexes between 0.18 nM Ang1 and cellular Tie2. ( a ) Time course of formation of Ang1 bound to one (A3M; purple), two (A2M2; blue), three (A1M3; red) and four (AM4; black) surface receptors. Amount of complex is shown in log scale (arbitrary units). ( b ) Time course of the formation of Ang1 fully complexed with four cell surface receptors (AM4). ( c ) Time course of formation of AM4 at different Ang1 concentrations up to 1.8 nM. Increasing Ang1 concentration is indicated as an inverted triangle. ( d ) Dependence of AM4 formation on Ang1 (A4) concentration.

Article Snippet: Recombinant human Angpt1, antibodies recognizing Tie2 extracellular domain and anti-phosphoY 992 -Tie2 antibody were obtained from R&D Systems.

Techniques: Binding Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Regulation of Angiopoietin Signalling by Soluble Tie2 Ectodomain and Engineered Ligand Trap

doi: 10.1038/s41598-017-03981-6

Figure Lengend Snippet: Numerical simulations of the effects of sTie2 on Ang1 binding. ( a ) Time course of formation of Ang1 bound to one (A3S; purple), two (A2S2; blue), three (A1S3; red) and four (AS4; black) soluble Tie2 ectodomains. Initial Ang1 concentration 0.18 nM, initial sTie2 concentration 1 nM. Amount of complex is shown in log scale (arbitrary units). ( b ) Time course of the formation of Ang1 complexed with a single sTie2 (A3S). ( c ) Time course of effects of different sTie2 concentrations up to 150 nM on free Ang1 (A4) concentration starting with a free Ang1 concentration of 0.18 nM ( d ) Concentration-dependence of sTie2 effects on free Ang1 (A4) concentration. ( e ) Time course of the effects of increasing sTie2 concentrations on the ability of 0.18 nM Ang1 to form the signalling-competent AM4 complex at the cell surface. ( f ) Concentration-dependence of sTie2 effects on formation of signalling-competent AM4 complex at the cell surface.

Article Snippet: Recombinant human Angpt1, antibodies recognizing Tie2 extracellular domain and anti-phosphoY 992 -Tie2 antibody were obtained from R&D Systems.

Techniques: Binding Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Regulation of Angiopoietin Signalling by Soluble Tie2 Ectodomain and Engineered Ligand Trap

doi: 10.1038/s41598-017-03981-6

Figure Lengend Snippet: Effects of sTie2 on Ang1 activation of cell surface Tie2. ( a ) Coomassie-stained gel showing purified monomeric sTie2 used in experiments, mass markers are indicated in kDa. ( b ) Effects of sTie2 on Ang1 activation of cellular Tie2. HUVEC were stimulated with 0.18 nM Ang1 for 15 minutes in the presence of monomeric sTie2 concentrations shown. Cells were lysed and activated cellular Tie2 detected with anti-phosphoY 992 -Tie2 immunoblotting. Blots were stripped and probed with anti-Tie2 to quantify loading of tracks. ( c ) Blots from three independent experiments assessing cellular Tie2 activation were quantified by densitometric scanning and presented as mean and SEM for the effects of sTie2 on cellular Tie2 activated with Ang1. ( d ) Coomassie-stained gel showing purified dimeric engineered sTie2 used in experiments, mass markers are indicated in kDa. ( e ) Effects of dimeric sTie2 on Ang1 activation of cellular Tie2. HUVEC were stimulated with 0.18 nM Ang1 for 15 minutes in the presence of dimeric sTie2 concentrations shown. Cells were lysed and activated cellular Tie2 detected with anti-phosphoY 992 -Tie2 immunoblotting. Blots were stripped and probed with anti-Tie2 to quantify loading of tracks. ( f ) Blots from four independent experiments assessing cellular Tie2 activation were quantified by densitometric scanning and presented as mean and SEM for the effects of sTie2 on cellular Tie2 activated by Ang1. Images of blots have been cropped for clarity.

Article Snippet: Recombinant human Angpt1, antibodies recognizing Tie2 extracellular domain and anti-phosphoY 992 -Tie2 antibody were obtained from R&D Systems.

Techniques: Activation Assay, Staining, Purification, Western Blot

Journal: eLife

Article Title: Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation

doi: 10.7554/eLife.64909

Figure Lengend Snippet: ( a ) Using flow cytometry, CEC populations were gated from peripheral blood mononuclear cells (PBMCs) using a strategy involving positive (nuclear stain and CD31) and negative (CD45 and CD133) markers before characterization with three separate markers of endothelial activation. ( b ) and ( c ) Scatterplot visualization of the number of CECs and endothelial progenitor cells (EPCs) per million PBMCs identified from each sample with mean and standard error of mean for each group shown. ( d ) and ( e ) Boxplots extending from 25th to 75th percentile with bar showing mean and whiskers indicating the minimum and maximum number of CECs per million PBMCs from each group staining positive for endothelial activation markers ICAM1, SELP, or CX3CL1. Kruskal—Wallis test was performed ( b—e ) to test for difference between the groups with Dunn’s multiple comparison test carried out for pairwise testing post hoc. ( f ) and ( g ) Cumulative analysis of patient frequency data of CECs staining positive for endothelial activation markers, ICAM1, SELP, and/or CX3CL1. Chi-squared goodness-of-fit test was performed to assess for difference in frequencies between the groups in (f) and (g).

Article Snippet: AF488-conjugated monoclonal antibody against human CX3CL1/fractalkine chemokine domain , IC365G-100UG (Research And Diagnostic Systems, Inc (R&D Systems, Inc), Minneapolis, Minnesota, United States.

Techniques: Flow Cytometry, Staining, Activation Assay

Journal: eLife

Article Title: Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation

doi: 10.7554/eLife.64909

Figure Lengend Snippet:

Article Snippet: AF488-conjugated monoclonal antibody against human CX3CL1/fractalkine chemokine domain , IC365G-100UG (Research And Diagnostic Systems, Inc (R&D Systems, Inc), Minneapolis, Minnesota, United States.

Techniques: Fluorescence, Flow Cytometry, Diagnostic Assay, Luminex, Software, Multiplex Assay, Functional Assay

Journal: eLife

Article Title: Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation

doi: 10.7554/eLife.64909

Figure Lengend Snippet:

Article Snippet: AF488-conjugated monoclonal antibody against human CX3CL1/fractalkine chemokine domain , IC365G-100UG (Research And Diagnostic Systems, Inc (R&D Systems, Inc), Minneapolis, Minnesota, United States.

Techniques: Diagnostic Assay