Cd54 icam 1

HRMEC were seeded in 10 cm² dishes and were placed in serum-reduced medium at 85% confluence. Cells were then treated with vehicle or 1 ng/ml TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM) for 4 hours. Cells were then washed twice in cold PBS and lysed using radio-immunoprecipitation assay (RIPA) buffer (Qiagen) with protease inhibitors (Roche; Basel, Switzerland). Samples were equilibrated for total protein concentration using a Pierce BCA assay, subjected to 10% SDS-PAGE, and gels were transferred to nitrocellulose membranes using the iBlot system (Thermo Fisher Scientific). Membranes were blocked and probed in 5% BSA for CD54/ICAM-1 (1:1000; Cell Signaling) and β-Actin (1:4000; Thermo Fisher Scientific) or 5% milk for VCAM-1 (1:1000; Abcam; Cambrdige, UK). Blots were then labeled with horseradish peroxidase-conjugated secondary antibodies (1:2000). β-Actin was used as a loading control. Membranes were incubated in Pierce ECL Western blotting substrate and developed with a ChemiDoc MP (Bio-Rad; Hercules, CA). Blots were then quantified using ImageJ software.