Anti caveolin 1 antibody

Control and treated cells were harvested, washed in cold PBS, lysed by freeze-thaw in a lysis buffer followed by measuring proteins using a Bio-Rad Protein Assay Kit. Approximately 40-μg of protein was applied to each lane and subjected to electrophoresis in SDS-PAGE (10%), followed by immunoblot analyses on the polyvinylidene fluoride (PVDF) membranes, as previously described by Albert et al [24 (link)]. The PVDF membranes were incubated overnight (6–10°C) with anti-5-LOX antibody or BLT1 receptor polyclonal antibody (rabbit polyclonal, Cayman Chemical, Ann Arbor, MI), anti-caveolin-1 antibody (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, monoclonal, Cell Signaling, Danvers, MA). The membranes were washed with 0.05% Tween-20 in Tris-Buffered saline (TBST) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Thermo Scientific, Rockford, IL) and/or goat anti-rabbit IgG (KPL Biomedical, Chantilly, VA). An enhanced chemi-luminescence (ECL) technique was used for the detection of protein bands. The intensities of protein bands were measured using Labworks software (UVP Lab Products, Upland, CA).

HUVECs were obtained from Cell Applications (San Diego, CA) and were maintained and exposed to shear stress as previously described^{32 (link)}. Briefly, a circulating flow system was used to impose atheroprotective pulsatile shear stress (PS, 12 ± 4 dyn/cm²) or atheroprone oscillatory shear stress (OS, 1 ± 4 dyn/cm²). The flow system was held at 37°C and ventilated with 95% humidified air plus 5% CO₂. Antibodies against H3K4me1 and H3K27ac were from Active Motif (Carlsbad, CA); anti-eNOS, anti-phospho-eNOS (Ser1177), and anti-calmodulin antibodies were from Abcam (Cambridge, MA); anti-α-tubulin, anti-phospho-eNOS (Thr495), and horseradish peroxide-conjugated anti-rabbit and anti-mouse antibodies were from Cell Signaling Technology (Beverly, MA); anti-caveolin-1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The atorvastatin hemicalcium salt was from Tocris Biosciences (Bristol, UK).