Anti lrp

The endothelial cells were lysate with a T-PER^® Tissue Protein Extraction Reagent (Thermo Scientific) adding Halt^TM protease & Phosphatase inhibitor cocktail (1:100, Thermo Scientific). Cells were centrifuged at 13000rpm at 4°C for 20 min. Pierce^® BCA protein assay kit was used for detecting concentration of protein as followed manufacture's protocol (Thermo Scientific). Samples were mixed with 5× sample buffer (BIOSESANG, INC. Seongnam, South Korea) and boiled at 95°C for 5 min. Proteins were transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Bedford, MA, USA). After blocking with 5% bovine serum albumin (BSA), membranes were incubated with the anti-LRP (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NF-κB p65 (1:500, Santa Cruz Biotechnology), anti-Cox-2 (1:500, Santa Cruz Biotechnology), anti-Bax (1:1000, Merck Millipore, Bedford, MA, USA), anti-Bcl-2 (1:1000, Abcam, Cambridge, UK) primary antibodies respectively. Horseradish peroxidase-conjugated anti-goat, anti-mouse, or anti-rabbit IgG reagents (1:5000) were used as secondary antibodies. The β-actin (1:5000, Santa Cruz Biotechnology) was used as an internal control. The bands were visualized with enhanced chemiluminescence reagents (ECL Plus; Amersham Biosciences, Piscataway, NJ, USA) under the LAS 4000 program (GE Healthcare, Pittsburgh, PA, USA).