2.4. Western Blot Analysis

C- and GD-HUVECs were stimulated as described in the experimental protocol, and Western blot analysis was performed as previously described [10]. For the specific experiment, cells were lysed and 30 μg total protein was resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted using mouse monoclonal anti-VCAM-1 and ICAM-1 (1 : 1000 and 1 : 500, respectively) and mouse monoclonal anti-β-actin (1 : 10.000). The membranes were then incubated with peroxidase-conjugated secondary antibodies (1 : 10.000). Band densities of proteins were detected and quantified by using the Alliance Chemiluminescence Imaging System (UVItec Limited, Cambridge, United Kingdom). Densities of VCAM-1 and ICAM-1 proteins were divided by those of β-actin content, and the ratio was indicated as arbitrary units.