Journal: PLoS Computational Biology

Article Title: Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces

doi: 10.1371/journal.pcbi.1007207

Figure Lengend Snippet: ( a ) G6 and 18 low-energy designs, each encoding 4–10 mutations relative to G6 (number of mutations is indicated above the bars) were tested for binding using yeast display at 8 nM VEGF concentration, resulting in seven designs that showed comparable or higher binding signal compared to G6. G6 des1 and G6 des13 were chosen for further characterization (colored in blue and orange, respectively). ( b ) SPR kinetic analysis of VEGF binding with twofold dilutions from a maximal concentration of 100 nM by G6, G6 des1 , and G6 des13 Fabs demonstrated faster binding on-rate in the designs ( k a = 2.3 * 10 5 M -1 s -1 , 3.27 * 10 5 M -1 s -1 and 5.3 * 10 5 M -1 s -1 , respectively). G6 des13 also improved binding off-rate ( k d = 3.2 * 10 −5 s -1 compared to 6 * 10 −5 s -1 in G6), resulting in an improved dissociation constant ( K D = 60 pM compared to 270 pM in G6). ( c & d ) Thermal denaturation and temperature of aggregation onset experiments, respectively, using microscale thermophoresis indicated substantially higher apparent stability in the designs. ( e ) Computational mutation-tolerance mapping indicated 11 positions at the vL-vH interface of the anti-VEGF antibody G6 (spheres) with potentially tolerated mutations. Thumbnails indicate selected mutations in a model structure of G6 des13 relative to G6 (gray). ( f ) Expression levels in HEK293 cells of G6 and the designs formatted as IgG were measured using Western blot analysis showing approximately an order of magnitude improvement in IgG expression levels for the designs. (g) Native mass-spectrometry analysis exhibited higher tolerance to applied collision energy in G6 des13 compared to G6, both formatted as IgG. The error bars represent standard deviations inferred from three repeats.

Article Snippet: The wild-type and designed antibodies were tested for binding by flow cytometry with 8 nM biotinylated VEGF (Recombinant Human VEGF 165, Biotinylated Protein R&D systems).

Techniques: Binding Assay, Concentration Assay, Microscale Thermophoresis, Mutagenesis, Expressing, Western Blot, Mass Spectrometry

Journal: Oncotarget

Article Title: DSGOST inhibits tumor growth by blocking VEGF/VEGFR2-activated angiogenesis

doi: 10.18632/oncotarget.7982

Figure Lengend Snippet: (A) HUVECs were pretreated with DSGOST at different concentrations for 60 minutes and then treated with VEGF (50 ng/ml) for another 60 minutes. (B) Cells were treated with VEGF and DSGOST for the indicated time points. (C) DSGOST and btVEGF were treated on the plate where recombinant human VEGFR2 was coated.

Article Snippet: The plate was washed 3 times and added with 100 μl of diluted standards (biotinylated VEGF (btVEGF), R & D systems, Minneapolis, USA) or compounds (with 50 ng/ml btVEGF) in PBS.

Techniques: Recombinant

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 1. Effect of 17-estradiol and progesterone on rhVEGF165 bind- ing to human myometrial MEC. A, Flow cytometry traces showing that (left panel) proliferating MEC bind more rhVEGF165 than qui- escent MEC (basal binding) and (right panel) 17-estradiol (E2), but not progesterone (P4) increased basal rhVEGF165 binding. The neg- ative control represents nonspecific binding. B, Quiescent MEC in- cubated with vehicle, 10 nmol/liter E2, 100 nmol/liter progesterone (P), and 17-estradiol progesterone for 18 h at 37 C in the presence 10 g/liter VEGF were harvested and rhVEGF165 binding measured. Results are mean SEM from four experiments on separate MEC isolates. *, P 0.01.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Binding Assay, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 2. 17-estradiol increased rhVEGF165 binding to myometrial MEC in a time- and dose-dependent manner. A, Time course of rh- VEGF165 binding to MEC incubated with 10 nmol/liter 17-estradiol. B, Concentration-response curve of MEC incubated with or without 17-estradiol for 18 h at 37 C. MFI of rhVEGF165 binding is reported as percentage of control (vehicle-treated cells). Results are mean

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Concentration Assay, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 5. 17-estradiol increases VEGFR-2 expression in ER ex- pressing but not in ER myometrial MEC. Quiescent MEC in hu- man serum-free medium were incubated for 18 h at 37 C in the presence of 0 (vehicle), 1 and 10 nmol/liter 17-estradiol and rhVEGF165 binding (f) and VEGFR-2 expression () then measured by flow cytometry. A, Representative flow cytometry histograms of VEGFR-2-FITC fluorescence for ER and ER samples. Negative control is mouse IgG1. B, ER expressing MEC. C, ER MEC. Results are expressed as percentage of control (vehicle only) rhVEGF165 binding or MFI of VEGFR-2 expression. Means SEM from n 4 (B) and n 6 (C) separate MEC isolates. *, P 0.05; **, P 0.01.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Incubation, Binding Assay, Flow Cytometry, Fluorescence, Negative Control, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 6. Antiestrogen ICI 182,780 inhibits 17-estradiol up-regula- tion of rhVEGF165 binding to myometrial MEC and 17-estradiol enhancement of VEGF-stimulated MEC proliferation. A, Quiescent MEC in serum-free medium containing 10 g/liter VEGF were incu- bated for 18 h at 37 C with either 0.1 or 10 nmol/liter 17-estradiol in the presence or absence of 0.01 or 1 mol/liter ICI 182,780 respec- tively and rhVEGF165 binding was then measured. Results are mean SEM from three experiments on separate MEC isolates. *, P 0.01; **, P 0.05. B, Quiescent myometrial MEC (4 103) seeded in triplicate were incubated with 1 mol/liter ICI 182,780 in the pres- ence or absence of 10 nmol/liter 17-estradiol for 6 d at 37 C in phenol-red free M199 medium containing 2 g/liter VEGF and 5% ch-HS and 15% ch-FCS, then MTS reagent added and absorbance measured. Results are presented as the change in absorbance over 6 d expressed as percentage of control MEC (vehicle only). Means ( SEM) from five experiments on separate MEC isolates are shown. *, P 0.01; **, P 0.05.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Control