Human integrin receptors avb3 (R&D Systems, Minneapolis, MN, USA) and avb5 (EMD Millipore Corporation, Inc., Temecula, CA, USA) were diluted to 0.5 μg/mL in coating buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MnCl2, 2 mM CaCl2, and 1 mM MgCl2. An aliquot of diluted receptor (100 μL/well) was added to 96-well microtiter plates (Nunc MaxiSorp, Termo Fisher Scientific, Roskilde, DK) and incubated overnight at 4 °C. The plates were incubated with blocking solution (coating buffer plus 1% bovine serum albumin) for additional 2 h at room temperature to block nonspecific binding. After washing 2 times with blocking solution, plates were incubated shaking in the dark for 3 h at room temperature, with various concentrations (10^-5-10^-12 M) of test compounds in the presence of 1 μg/mL vitronectin (Molecular Innovations, Novi, MI, USA) biotinylated using an EZ-Link Sulfo-NHS-Biotinylation kit (Pierce, Rockford, IL, USA). After washing 3 times, the plates were incubated shaking for 1 h in the dark, at room temperature, with streptavidin-biotinylated peroxidase complex (Amersham Biosciences, Uppsala, Sweden). After washing 3 times with blocking solution, plates were incubated with 100 μL/well of Substrate Reagent Solution (R&D Systems, Minneapolis, MN, USA) for 30 min shaking in the dark, before stopping the reaction with the addition of 50 μL/well 2 N H2SO4. Absorbance at 415 nm was read in a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc.). Each data point represents the average of triplicate wells; data analysis was carried out by nonlinear regression analysis with GraphPad Prism software. Each experiment was repeated in duplicate.

Human integrin receptors avb3 (R&D Systems, Minneapolis, MN, USA) and avb5 (EMD Millipore Corporation, Inc., Temecula, CA, USA) were diluted to 0.5 μg/mL in coating buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MnCl2, 2 mM CaCl2, and 1 mM MgCl2. An aliquot of diluted receptor (100 μL/well) was added to 96-well microtiter plates (Nunc MaxiSorp, Termo Fisher Scientific, Roskilde, DK) and incubated overnight at 4 °C. The plates were incubated with blocking solution (coating buffer plus 1% bovine serum albumin) for additional 2 h at room temperature to block nonspecific binding. After washing 2 times with blocking solution, plates were incubated shaking in the dark for 3 h at room temperature, with various concentrations (10^-5-10^-12 M) of test compounds in the presence of 1 μg/mL vitronectin (Molecular Innovations, Novi, MI, USA) biotinylated using an EZ-Link Sulfo-NHS-Biotinylation kit (Pierce, Rockford, IL, USA). After washing 3 times, the plates were incubated shaking for 1 h in the dark, at room temperature, with streptavidin-biotinylated peroxidase complex (Amersham Biosciences, Uppsala, Sweden). After washing 3 times with blocking solution, plates were incubated with 100 μL/well of Substrate Reagent Solution (R&D Systems, Minneapolis, MN, USA) for 30 min shaking in the dark, before stopping the reaction with the addition of 50 μL/well 2 N H2SO4. Absorbance at 415 nm was read in a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc.). Each data point represents the average of triplicate wells; data analysis was carried out by nonlinear regression analysis with GraphPad Prism software. Each experiment was repeated in duplicate.