Incubation proceeded for 1 h at 37°C. After three washings with PBS – T, 100 μL of a 1:5,000 dilution of HRP – conjugated goat anti – mouse IgG (Sigma) in PBS was added per well for 1 h at 37°C. The wells were washed three times, and o – phenylenediamine (OPD) (1 mg/mL) in citrate phosphate buffer (pH 5.0) plus 1 μL/mL H2O2 was added (100 μL per well). The reaction proceeded for 10 min and was interrupted by the addition of 50 μL of 4 N H2SO4. The absorbance at 492 nm was determined in a microplate reader (TP – reader, Thermo). For statistical analyses, the binding of recombinant proteins to ECM macromolecules

A-1210477 chemical structure and to serum components were compared to its binding to gelatin and by Student’s two – tailed t test. Metaperiodate treatment of XAV-939 in vitro laminin Microtitre wells were coated with 1 μg of laminin in 50 mM sodium acetate buffer, pH 5.0, and incubated for 16 h at

4°C. Wells were washed three times with the same buffer, and laminin was treated with different sodium metaperiodate concentrations (0–100 mM) in the same buffer for 15 min at 4°C in the dark. After three washes with 50 mM sodium find more acetate buffer, wells were blocked with 200 μl of 1% BSA for 1 h at 37°C. Binding of recombinant proteins (1 μg in PBS per well) to periodate – treated laminin was evaluated as described above. Dose–response curves First, 96 – well plates were coated overnight in PBS at 4°C with 100 μl of 10 μg/ml PLG, laminin or C4bp. Plates were then blocked and increasing concentrations of the purified recombinant proteins (0–6 μM) were added (100 μl/well in PBS). The assessment of bound proteins was performed by incubation

for 1 h at 37°C with the antiserum raised against each protein at the dilution of 1:750, followed by HRP – conjugated goat anti-mouse IgG (Sigma) (1:5,000 in PBS). The reactions were detected with OPD as describe above. The ELISA data were used to calculate the dissociation constant (KD) according to the method described by tuclazepam Pathirana et al. [60] and Lin et al. [61], based on the equation: , where A is the absorbance at a given protein concentration, Amax is the maximum absorbance for the ELISA plate reader (equilibrium), [protein] is the protein concentration and KD is the dissociation equilibrium constant for a given absorbance at a given protein concentration (ELISA data point). Plasmin enzymatic activity assay 96 – well ELISA plates were coated overnight with 10 μg/ml recombinant proteins in PBS at 4°C. Lsa63, which does not bind PLG [21] and BSA were employed as negative control. Plates were washed once with PBS – T and blocked for 2 h at 37°C with PBS with 10% (wt/vol) non – fat dry milk. The blocking solution was discarded and 100 μl/well of 10 μg/ml human PLG was added, followed by incubation for 2 h at 37°C. Wells were washed three times with PBS – T, and then 4 ng/well of human uPA (Sigma – Aldrich) were added.