Journal: Molecular Oncology

Article Title: Focal adhesion kinase inhibitors are potent anti‐angiogenic agents

doi: 10.1016/j.molonc.2011.10.004

Figure Lengend Snippet: FAK inhibitors decrease number of viable endothelial cells in a dose‐dependent manner. HUVEC were incubated with various concentrations of either PF‐228 (A) or FI14 (B) in the presence of VEGF. Cells treated with vehicle (DMSO) were used ...

Article Snippet: Recombinant human vascular endothelial growth factor (VEGF) (rhVEGF 165 ; R&D Systems, Minneapolis, MN) was reconstituted according to the manufacturer's instructions.

Techniques: Incubation

Journal: Molecular Oncology

Article Title: Focal adhesion kinase inhibitors are potent anti‐angiogenic agents

doi: 10.1016/j.molonc.2011.10.004

Figure Lengend Snippet: FAK inhibitors induce cell cycle arrest and apoptosis in treated endothelial cells. Seeded HUVEC were treated with DMSO as vehicle control or varying concentrations of PF‐228 or FI14 in the presence of 50 ng/mL VEGF for 48 h. ...

Article Snippet: Recombinant human vascular endothelial growth factor (VEGF) (rhVEGF 165 ; R&D Systems, Minneapolis, MN) was reconstituted according to the manufacturer's instructions.

Techniques: Control

Journal: Molecular Oncology

Article Title: Focal adhesion kinase inhibitors are potent anti‐angiogenic agents

doi: 10.1016/j.molonc.2011.10.004

Figure Lengend Snippet: FAK inhibitors block HUVEC sprouting on collagen I gels. For assessing formation of sprouts, HUVEC were seeded onto collagen I gel‐coated plates and treated with 50 ng/ml VEGF in the absence (with added DMSO as a vehicle control) ...

Article Snippet: Recombinant human vascular endothelial growth factor (VEGF) (rhVEGF 165 ; R&D Systems, Minneapolis, MN) was reconstituted according to the manufacturer's instructions.

Techniques: Blocking Assay, Control

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