Enos antibody

Samples were lysed (20 mM Tris, 1% Deoxycholate, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM PMSF, 0.1% SDS, 1 μg/mL leupeptin, 50 mM mM NaF, 10% glycerol, Pierce complete protease inhibitor, pH 7.4) at 4 °C for 20 min, then centrifuged for 10 min at 10,000 g, 4 °C to remove insoluble material. Cell lysates were normalized for protein content, separated by SDS-PAGE on a 4–20% Bis–Tris gel (Lonza), and transferred to a nitrocellulose membrane (Invitrogen, Iblot). Membranes were blocked with 5% Blotto (Bioexpress) and incubated overnight with primary p-eNOS (1179) antibody (Invitrogen) or eNOS antibody (BD biosciences) at 4 °C. Secondary horseradish peroxidase-conjugated antibodies were detected with an enhanced chemiluminescence kit (Western Lightning, PerkinElmer) and visualized with a Fluorchem digital imager (Alpha Innotech). Band intensity was quantified using AlphaEase FC software and expressed as a ratio of p-eNOS/eNOS. For studies on phosphorylation after shear stress, results were normalized by the no flow condition for both cholesterol-enriched and unenriched cells.