Western Blot Analysis of Endothelial Cells

ECs were treated with AS for 24 h, and Western blot was performed according to a previously described method [25 (link)]. Briefly, ECs were harvested and lysed in protein-extraction solution (Intron Biotechnology, Inc., Kyunggi, Korea) containing protease inhibitors and phosphatase inhibitors for 10 min at 4 °C. The total protein concentration in the supernatants was measured using the Bradford assay [11 (link)]. After heating at 95 °C for 5 min, total protein samples (30 μg) were subjected to 6% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) at 100 V for 60 min to 100 min. The membranes were incubated with 5% bovine serum albumin in Tris-buffered saline with 0.05% Tween 20 (TBST) for 30 min at room temperature, and then with diluted primary antibodies (1:200–1:1000) in 5% BSA in TBST overnight at 4 °C. The membranes were washed three times with TBST and incubated with the corresponding secondary antibodies. Protein bands were detected using an enhanced chemiluminescence detection kit (Intron Biotechnology, Inc.) and a LAS-1000 Imager (Fuji Film Corp., Tokyo, Japan). Semi-quantitative analysis of densitometry results was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).