Journal: BMC Cancer

Article Title: Combining the receptor tyrosine kinase inhibitor AEE788 and the mammalian target of rapamycin (mTOR) inhibitor RAD001 strongly inhibits adhesion and growth of renal cell carcinoma cells

doi: 10.1186/1471-2407-9-161

Figure Lengend Snippet: Adhesion of RCC cells to extracellular matrix proteins . A498, Caki-1 or KTC-26 cells were treated with 1 μM AEE788 or 1 nM RAD001, applied alone or in combination. Non-treated cells served as the controls. Cells were then added to immobilized collagen, laminin, or fibronectin at a density of 0.5 × 10 6 cells/well for 60 min. Plastic dishes were used to evaluate unspecific binding (background control). Non-adherent tumor cells were washed off in each sample, the remaining cells were fixed and counted in five different fields (5 × 0.25 mm 2 ) using a phase contrast microscope. Mean values were calculated from the five counts. Specific adhesion capacity (background adhesion on plastic surface was subtracted from adhesion to matrix proteins) is depicted as adherent cells/mm 2 . One representative of six experiments is shown. * indicates significant difference to controls (p < 0.05).

Article Snippet: 6-well plates were coated with collagen G (extracted from calfskin, consisting of 90% collagen type I and 10% collagen type III; Seromed; diluted to 100 μg/ml in PBS), laminin (derived from the Engelbreth-Holm-Swarm mouse tumor; BD Biosciences; diluted to 50 μg/ml in PBS), or fibronectin (derived from human plasma; BD Biosciences; diluted to 50 μg/ml in PBS) overnight.

Techniques: Binding Assay, Microscopy

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: Plasma and recombinant VWF bind to fibronectin. Human fibronectin was captured on a plate and used to bind plasma VWF (healthy controls or subjects with type 3 VWD) or recombinant VWF. Decreased binding was noted for VWF constructs unable to form multimeric structures (2773R and 87S) as well as constructs with VWF A1 domain variants (1392A, 1395A, 1399H). Results are graphed as a ratio of VWF bound to fibrinogen over VWF antigen to account for minor differences in total protein. Error bars show 1 standard deviation. Results are average of ≥3 experiments. * signifies P <.05 and ** signifies P <.02 (plasma samples compared against each other, recombinant samples compared to WT recombinant VWF). VWD, von Willebrand disease; VWF, von Willebrand factor; VWF:Ag, von Willebrand factor antigen; WT, wild‐type

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Clinical Proteomics, Recombinant, Binding Assay, Construct, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: Fibronectin preferentially interacts with higher‐molecular‐weight VWF multimers. Binding of VWF to human fibronectin is shown using purified preparations of recombinant ultra‐high‐molecular‐weight VWF multimers (ultra), high‐molecular‐weight VWF multimers (high), medium‐molecular‐weight VWF multimers (medium), and low‐molecular‐weight VWF multimers (low). Results are graphed as a fraction of WT recombinant VWF binding with all multimers present to normalize for the total amount of VWF present in each preparation. Error bars show 1 standard deviation. Results are average of ≥3 experiments. VWF, von Willebrand factor; VWF:Ag, von Willebrand factor antigen; WT, wild‐type

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Molecular Weight, Binding Assay, Purification, Recombinant, High Molecular Weight, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: Inhibition of VWF‐fibronectin interactions. VWF binding to human fibronectin was measured alone (WT), and in the presence of anti‐VWF antibody AVW‐3, which blocks the A1 domain (anti‐VWF A1), a polyclonal VWF antibody (polyclonal anti‐VWF), collagen type III and IV, and extracellular matrix proteins thrombospondin, vitronectin, and laminin. Results are graphed as a percent of WT VWF bound to fibronectin in the absence of antibody. Error bars show 1 standard deviation. Results are average of three or more experiments. ** signifies P < .01. ECM, extracellular matrix; VWF, von Willebrand factor; VWF:Ag, von Willebrand factor antigen; WT, wild‐type

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Inhibition, Binding Assay, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: Binding affinities of VWF for collagen IV and fibronectin

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Binding Assay, Concentration Assay

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: VWF binding to fibronectin and collagen IV. VWF binding to human fibronectin (black) and human collagen IV (gray) was measured using the Octet Biosensor to determine binding affinities. The x axis shows varying VWF concentrations from 0 to 3 μg/mL, and the y axis shows the K D in nM. K D , dissociation constant; VWF, von Willebrand factor

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Binding Assay