Sc 1504 r

HUVEC were washed with PBS and lysed with a lysis buffer containing 0.5% SDS in 10 mM Tris–HCl (pH 7.4) and 1 mM ortovanadate. Protein concentration was measured by the BCA protein assay^™ and proteins were resolved by SDS-PAGE and electrotransferred onto Immobilon polyvinylidene diflouride membranes (Millipore). Western blot analysis was performed using antibodies against NOR-1 (clone 1E11, Abnova) or VCAM-1 (sc-1504-R, Santa Cruz Biotechnology). Detection was performed using the appropriate horseradish peroxidase-conjugated antibody (Dako) and a chemiluminescent detection system (Supersignal West Dura^™, Pierce). The size of detected proteins was estimated using protein molecular-mass standards (Fermentas). β-actin (ab8226, Abcam) was used as a loading control.

Equal amounts of protein were separated on a 10% SDS-polyacrylamide gel and transferred onto a 0.45 μm nitrocellulose membrane using a wet blot chamber (Bio-Rad, Hercules, USA). The blots were incubated overnight with the primary antibody at 4°C. Proteins were detected using Western-Bright ECL spray (Advansta, Menlo Park, USA) after incubation for 1 h at room temperature with the appropriate secondary antibody. Primary antibodies used: anti-ZO1 tight junction protein (abcam, ab59720; 1:100); anti-Vascular Cell Adhesion Molecule 1 (VCAM-1) (Santa Cruz, sc-1504-R; 1:500). Secondary antibody: anti-rabbit IgG, HRP-linked (GE-Healthcare, NA934; 1:5000).