Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Affinity maturation of the Ang2-BD RGD -based library bi-specific for α v β 3 integrin and Tie2-Fc. a Ang2-BD was presented on the yeast cell surface as a fusion with agglutinin proteins. Display levels were detected using primary antibodies against the C-terminal cMyc tag (chicken anti-cMyc antibodies) and phycoerythrin (PE)-conjugated anti-chicken antibodies. Binding to Tie2-Fc was determined using fluorescein isothiocyanate (FITC)-conjugated anti-human Fc antibodies. Binding to α v β 3 integrin was determined using FITC-labeled mouse anti-α v integrin antibodies. b – g FACS analysis of the binding of the bi-specific Ang2-BD-based library to α v β 3 integrin in different screening steps. Quadrant gate statistics are indicated in each panel b negative control. c Ang2-BD WT expression and α v β 3 integrin binding (10 nM). d Expression of the bi-specific Ang2-BD RGD -based library and α v β 3 integrin binding (10 nM) at pre-sorting and e – g expression of the bi-specific Ang2-BD-based library and α v β 3 integrin binding (10 nM) after sorts 1, 3, and 5, respectively. h Binding of isolated yeast-displayed bi-specific Ang2-BD RGD clones to Tie2 (20 nM). Data were normalized to the yeast surface expression levels of each clone and Tie2 binding of Ang2-BD WT . i Binding of isolated yeast-displayed bi-specific Ang2-BD RGD clones to α v β 3 , α 5 β 1 , α v β 5 , α 4 β 7 , α IIb β 3 , and α 3 β 1 integrins (50 nM). Data were normalized to the yeast surface expression level of each clone. Data shown represent the average of triplicates from independent experiments, and error bars represent the standard error of the mean

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Binding Assay, Labeling, Negative Control, Expressing, Isolation, Clone Assay

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Binding affinities and kinetic rate constants for binding of Ang2-BD BC5 to immobilized integrins

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Binding Assay

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Binding of Ang2-BD bi-specific variants to recombinant and cell-expressed human Tie2 and α v β 3 integrin. a Representative SPR sensorgram of Ang2-BD bi-specific variants (400 nM) binding to both α v β 3 integrin (immobilized) and soluble Tie2 (400 nM). Ang2-BD variants [ Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red)] were allowed to flow for 400 s followed by an additional 150 s flow of Tie2. Injection of running buffer followed by rhTie2 in IBB served as negative control (black). Injection steps are indicated on the sensorgrams. b Binding of Ang2-BD bi-specific variants to TIME cells. 1 × 10 5 cells were incubated with Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red) for 2 h at 4 °C with gentle agitation. Mean fluorescence values were determined by flow cytometry using fluorescently labeled antibodies against a FLAG epitope tag. “*” indicates a P value < 0.05 by one-way ANOVA upon comparing results between Ang2-BD variants at the same concentration. c Competitive binding of 1 μM Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red), alone or with a combination of 1000 ng/ml of FL-Ang1 (checkered bars), 10 μM cRGD (horizontally lined bars) and both 1000 ng/ml FL-Ang1 and 10 μM cRGD (vertically-lined bars). Mean fluorescence values were determined by flow cytometry using fluorescently labeled antibodies against a FLAG epitope tag. “*” indicates a P value < 0.05 by one-way ANOVA upon comparing results between Ang2-BD variants alone and with FL-Ang1 and cRGD competitors. Data shown represent the average of triplicates from independent experiments, and error bars represent the standard error of the mean

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Binding Assay, Recombinant, Injection, Negative Control, Incubation, Fluorescence, Flow Cytometry, Labeling, FLAG-tag, Concentration Assay

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Structure of the Tie2–Ang2-BD BC5 –α v β 3 complex. The Tie2 structure was aligned into the last frame of the Ang2-BD BC5 –integrin α v β 3 MD simulation. Tie2 is shown in cyan, Ang2-BD BC5 in beige, α v in yellow, and β 3 in green. Tie2-binding residues of Ang2 are colored magenta and NTCRGDCLP mutant residues in red. A zoom-in of the binding interface between Ang2-BD BC5 and α v β 3 integrin is also shown. On the right, an all-atom representation of the mutant residues of Ang2-BD BC5 is shown in stick form

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Binding Assay, Mutagenesis

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Inhibition of TIME cells adhesion and phosphorylation of Tie2, Akt, and FAK. a 5 × 10 4 TIME cells were incubated alone (cells only; black), with 500 ng/ml of FL-Ang1 (gray), or with a combination of 500 ng/ml FL-Ang1 and 1 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red) for 2 h on vitronectin-coated 96-well plates. b 5 × 10 4 TIME cells were incubated alone (cells only; black) or with 1 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red) for 2 h on vitronectin-coated 96-well plates. c For determining Tie2 phosphorylation, TIME cells were treated with control buffer (basal level; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 1 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red) for 15 min on vitronectin-coated 12-well plates. d Cell lysates were analyzed by Western blot using antibodies against phosphorylated Tie2 (pTie2), Tie2, and β-actin. e Akt phosphorylation was determined as in c except that the cells were treated with a combination of 500 ng/ml FL-Ang1 with 0.5 μM of the above proteins and the incubation time was 30 min. f Cell lysates were analyzed by Western blot using antibodies against phosphorylated Akt (pAkt), Akt, and β-actin. g FAK phosphorylation was determined as in c . h Cell lysates were analyzed by Western blot using antibodies against phosphorylated FAK (pFAK), FAK, and β-actin. “*” indicates a P value < 0.05 upon comparing the results between the cells-only control ( b ) and the tested proteins. “*” indicates a P value < 0.05 upon comparing the results between FL-Ang1 alone ( a , c , e , h ) and a combination of FL-Ang1 with the tested proteins. “&” indicates a P value < 0.05 upon comparing the results between cRGD ( a , b , c , e , h ) and the tested proteins. “#” indicates a P value < 0.05 upon comparing the results between Ang2-BD WT ( a , b , c , e , h ) and the tested proteins

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Inhibition, Incubation, Western Blot

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Endothelial cell tube formation and inhibition of cell viability by Ang2-BD bi-specific variants. a 3.25 × 10 4 TIME cells were treated with the indicated proteins overnight and washed, and pictures were taken using a × 2 objective. Scale bar, 500 μm. b Images of tube structures were analyzed for the number of generated junctions and the number of meshes in control buffer (cells only; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 1 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red). c Cell viability was determined by the XTT assay for control buffer (cells only; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 2 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red). “*” indicates a P value < 0.05 upon comparing the results between FL-Ang1 and the tested proteins. “&” indicates a P value < 0.05 upon comparing the results between cRGD and the tested proteins. “#” indicates a P value < 0.05 upon comparing the results between Ang2-BD WT and the tested proteins ( b , c )

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Inhibition, Generated, XTT Assay

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Inhibition of endothelial cell invasiveness by Ang2-BD bi-specific variants. a TIME cells were treated with the indicated proteins in Boyden chambers. Scale bar, 200 μm. b The invading cells accumulating at the bottom of the membrane were counted in 16 frames for each membrane and analyzed for the number of cells for control buffer (cells only; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 1 μM cRGD (purple), Ang2-BD WT (blue), cRGD together with Ang2-BD WT (orange), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red). “*” indicates a P value < 0.05 upon comparing the results between FL-Ang1 and the tested proteins. “&” indicates a P value < 0.05 upon comparing the results between cRGD and the tested proteins. “#” indicates a P value < 0.05 upon comparing the results between Ang2-BD WT and the tested proteins. “$” indicates a P value < 0.05 upon comparing the results between cRGD + Ang2-BD WT and the tested proteins. The data in Figs. , , and 6 were analyzed by one-way ANOVA. Data shown represent the average of triplicates from independent experiments, and error bars represent the standard error of the mean

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Inhibition

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Equilibrium binding affinities and kinetic rate constants for binding of Ang2-BD variants to immobilized Tie2 and α v β 3 integrin

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Binding Assay

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Sequencing analysis of isolated clones from the bi-specific Ang2-BD RGD library

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Sequencing, Isolation, Clone Assay

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Inhibition percentage of capillary tube formation, viability, and invasiveness of endothelial TIME cells growing on Matrigel vs FL-Ang1-treated controls

Article Snippet: The binding interactions of Tie2 to Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , and Ang2-BD BC10 were analyzed (Proteomics Unit, NIBN, BGU) by SPR using a ProteOn XPR36 instrument (Bio-Rad) as previously described [ ].

Techniques: Inhibition

Journal: Turkish Journal of Biochemistry

Article Title: Two approaches for measurement uncertainty estimation: which role for bias? Complete blood count experience

doi: 10.1515/tjb-2020-0357

Figure Lengend Snippet: Figure 1: Measurement uncertainty of PLT of four autoanalyzers with two different guideline, with contribution rates of RMSbias, u (cref) and imprecision of IQC. AACB, Australasian Association of Clinical Biochemists Uncertainty of Measurement Working Group Measurement Uncertainty Guideline; Nordtest, Handbook for Calculation of Measurement Uncertainty in Environmental Laboratories Guideline; u (Cref), uncertainty of external quality assurance sample; RMSbias, root mean square bias; IQC CV, imprecision of internal quality control; R1 and R2, two Beckman Coulter LH-780 autoanalyzers run in routine laboratory; E1 and E2, two Beckman Coulter DXH-800 autoanalyzers run in emergency laboratory.

Article Snippet: EQA program was BIO-RAD External Quality Assurance Services Hematology Program.

Techniques: Control

Journal: Turkish Journal of Biochemistry

Article Title: Two approaches for measurement uncertainty estimation: which role for bias? Complete blood count experience

doi: 10.1515/tjb-2020-0357

Figure Lengend Snippet: Figure 2: Measurement uncertainty of WBC of four autoanalyzers with two different guideline, with contribution rates of RMSbias, u (cref) and imprecision of IQC. AACB, Australasian Association of Clinical Biochemists Uncertainty of Measurement Working Group Measurement Uncertainty Guideline; Nordtest, Handbook for Calculation of Measurement Uncertainty in Environmental Laboratories Guideline; u (Cref), uncertainty of external quality assurance sample; RMSbias, root mean square bias; IQC CV, imprecision of internal quality control; R1 and R2, two Beckman Coulter LH-780 autoanalyzers run in routine laboratory; E1 and E2, two Beckman Coulter DXH-800 autoanalyzers run in emergency laboratory.

Article Snippet: EQA program was BIO-RAD External Quality Assurance Services Hematology Program.

Techniques: Control