Anti sr bi

Human macrophages were grown in culture flasks at a density of 1 × 10^7/mL for 12 h, washed twice with PBS, and then incubated in DMEM containing 10% autologous human serum. The non-targeting control siRNA, LXRα siRNA and LXRβ siRNA were added to the culture flasks separately, and cultured for 96 h. Cells were then harvested and protein extracts prepared in accordance with the manufacturer’s instructions. The proteins were then subjected to immunoblot analysis [12% SDS-PAGE; 60 μg protein per lane] using a rabbit anti-ABCA1, anti-ABCG1, anti-SR-BI, anti- LXRα, anti-LXRβ or anti-GAPDH (Abcam, USA)-specific antibody. Proteins were visualized using Enhanced Chemiluminescence reagents.

Total cell lysate was prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS and a mixture of protease inhibitors). Western blot analysis was performed using the same amount of protein. Protein concentration was determined by the Bradford (Sigma-Aldrich, St. Louis, MO, USA) method and equal quantities were subjected to Western blot analysis. SDS-PAGE-separated proteins were electroblotted onto a nitrocellulose membrane. The blots were incubated overnight at 4 °C with the following primary antibodies: anti-ERRα (ab76228; 1:1000); anti-SR-BI (ab52629; 1:1000); anti-HMGCR (ab215365; 1:500); from Abcam, Cambridge, UK; anti-Cyclin D1 (sc-8396; 1:500), anti-PARP-1 (sc-7150; 1:2000) and anti-GAPDH (sc-32233; 1:10,000) from Santa Cruz Biotechnology, Dallas, TX, USA. Anti βactin (ab8226; 1:1000; Abcam) was used as a loading control. All antibodies were incubated with appropriate horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were detected by the ECL Western blotting detection system (Santa Cruz Biotechnology, sc-2048). All the whole western blot figures can be found in the Supplementary Materials.