OPN was mixed with either AOM1

or control antibody. Antibody concentrations see more were titrated from 10 μM in a three-fold dilution series to approximately 0.1 nM. Human OPN and test antibody were pre-incubated for 1 hour at room temperature on a rotary mixer before being applied to the αVβ3 coated ELISA plates. After a washing step (3 times with Buffer 1 + 0.05% Tween-20 and three times with Buffer 1 alone), rabbit polyclonal anti-human OPN antibody (O-17, IBL, Japan) was added to the plates (100 μl/well) at a concentration of 4 μg/ml for 1 hour at room temperature. Plates were then washed (3 times with Buffer 1 + 0.05% Tween-20 and 3 times with Buffer 1 alone) and goat-anti-rabbit antibody (Fc specific) HRP conjugate (Jackson Immunoresearch, PA) was added to each well (100 μl/well, 1 in 5000 dilution in Block Buffer) for 1 hour at room temperature. Following final washes (3 times with Buffer 1 + 0.05% Tween-20 and 3 times with Buffer 1 alone) ELISA was developed with 100 μl/well

BM Blue POD substrate (Roche, NJ) and the AZD5363 nmr colorimetric reaction was stopped with 100 ul/well 0.2 M H2SO4. Absorbance at 450 nm was measured using a Spectromax plate reader (Molecular Devices, CA) and analysis was conducted using Microsoft Excel Data-Analysis Add-In fitting IC50 curves to a 4-paramter sigmoidal saturation binding model. Selectivity of AOM1 for OPN EIA/RIA plates (Corning, NY) were coated with 1 mg/ml of RGD-motif containing Ponatinib protein which included OPN, Thrombospondin, Vitronectin, ColIAI or Fibronectin (R&D Systems, MN) in Buffer 1 (PBS pH 7.2 containing 2 mM MgClR2R and 0.2 mM MnClR2R for 16 hours at 4°C). Plates were washed three times with Buffer 1 and were blocked with commercially available Blocking buffer (3% BSA (Rockland, PA) in Buffer 1) followed by washing three times with Buffer 1 and AOM1 was added at 0, 0.1, 1, 10, and 1000 nM in blocking buffer, and incubated at RT for 1 hr. Plates were washed (3 times with Buffer 1 + 0.05% Tween-20 and three times with

Buffer 1 alone). Goat Anti-Human IgG (Fc) Peroxidase Conjugate (Jackson Immunoresearch, PA) was added (1 in 5000 in block buffer) and plates were incubated at RT for 1 h followed by a wash (3 times with Buffer 1 + 0.05% Tween-20 and three times with Buffer 1 alone). BM Blue Solution (Roche, NJ) was used to develop the assay and quenched with 0.18 M HR2RSOR4R. Absorbance at 450 nm was detected using a Spectramax plate reader (Molecular Devices, CA) and data were analyzed using Microsoft Excel. Characterization of AOM1 Fab binding to OPN Binding of Fab fragment of AOM1 to recombinant OPN was determined using surface plasmon resonance (SPR) analysis on a Biacore 3000 instrument (GE Healthcare, CA).