The residue was dissolved in 50 mM of Tris-HCl (pH 7.4) and centrifuged at 20,000 rpm for 30 min. The supernatant was filtered through a 0.22-��m filter. The filtrate was subjected to fast protein liquid chromatography (FPLC; GE Healthcare UK Ltd. Buckinghamshire, England) separation Axitinib melanoma on HiTrap Q HP columns (5 mL; GE Healthcare). The columns were equilibrated with 50 mM of Tris-HCl (pH 7.4). The samples were then injected onto the columns, which were washed with the same buffer and eluted at a flow rate of 4 mL/min using a linear gradient consisting of 0�C2 M NaCl in 50 mM Tris-HCl (pH 7.4) over 45 min. The SRPX2 protein-containing fractions were then performed using gel-filtration chromatography (Superdex200 column, 16 mm��60 mm; GE Healthcare).
Expression constructs and purification of SRPX2-HA/His protein The method for producing the expression constructs was previously described [5]. Empty and SRPX2-HA/His vectors were then transfected into HEK293 cells using FuGENE6 transfection reagent (Roche Diagnostics, Basel, Switzerland), and the cells were then selected with hygromycin. The stable transfectant HEK293 cells were designated as HEK293-Mock and HEK293-SRPX2-HA/His. The conditioned medium of the HEK293-Mock and HEK293-SRPX2-HA/His cells was subjected to FPLC loading at 3 mL/min on a 5-mL HisTrap HP column (GE Healthcare). The bound protein was washed with 15 mL of wash buffer (WB: 50 mM Na2HPO4, 10 mM Tris-HCl, 20 mM imidazole [pH 8.0] and 600 mM NaCl,) and eluted in elution buffer (EB: WB+230 mM imidazole).
The SRPX2-HA/His protein-containing fractions were applied to an FPLC Superdex200 column (16 mm��60 mm; GE Healthcare) equilibrated with 0.15 M of ammonium bicarbonate. Elution was carried out using the same buffer at a flow rate of 1 mL/min. The SRPX2-HA/His-containing fractions were verified using western blotting and lyophilized. Digestion of SRPX2 by specific GAG-degrading enzymes Purified SRPX2-HA/His protein was digested with several specific enzymes including chondroitinase ABC and chondroitinase AC II (0.1 units in 40 mM Tris-HCl, 40 mM sodium acetate [pH 8.0] at 37��C for 2 h), chondroitinase B (0.02 units in 20 mM Tris-HCl, 0.25 ��M calcium acetate [pH 7.5] at 37��C for 2 h), heparinase I and heparinase II (0.05 units in 5 mM calcium acetate, 50 mM sodium acetate [pH 7.0] 37��C for h), keratanase (0.1 units in 7.5 ��M Tris-HCl [pH 7.
4] at 37��C for 2 h), and hyaluroinidase (0.02 M acetate buffer, 0.15 M NaCl [pH 6.0] at 60��C for 2 h). Enzymes were purchased from Seikagaku Kogyo. The samples were then analyzed using western blotting. Binding Assays An IAsys resonant mirror biosensor (Affinity Sensors, Dacomitinib Cambridge, UK) with a carboxymethyl dextran-sensing cuvette was used to determine the kinetic constants of hepatocyte growth factor (HGF) binding to immobilized SRPX2-HA/His.