P selectin

PBMC adhesion to ECs was measured as described.^{15 (link)} Briefly, HUVECs were incubated with M199 (Gibco, Grand Island, NE) containing 10% fetal bovine serum (FBS; Hyclone/Thermo Scientific, Waltham, MA) with or without recombinant human tumor necrosis factor α (TNFα, 10 ng/ml, Peprotech, Rocky Hill, NJ) for 4 h. In some treatment groups, the endothelial monolayer was preincubated with blocking antibodies for E-selectin or P-selectin (10 μg/mL, Biolegend, San Jose, CA) for 15 min at 37°C. At the end of incubation, the culture supernatant was removed and cells were washed 3 times with M199. PBMCs from Ldlr^−/− or DK mice were treated with or without 1 μM t-TUCB and 1 μM 14,15-EET for 6 h. Fluorescently labeled PBMCs (BCECF-AM, Invitrogen, Carlabad, CA) were incubated with HUVECs at 37°C for 1 h. After washing, PBMC adhesion to HUVECs was quantified by measuring fluorescent cells in areas of 5 independent images per condition. The graph depicts the mean of 3 independent experiments.

Commercial Abs used are: β-actin (Sigma-Aldrich, St. Louis, MO), Bcl2 (eBioscience, San Diego, CA), Bcl-xl (Abcam, Cambridge, MA), BAX (Santa Cruz Biotechnology, Santa Cruz, CA), HO-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and adhesion molecule ICAM-1, VCAM-1, PECAM-1, E-selectin, P-selectin (BioLegend, San Diego, CA).