Methods Binding and transactivation assays

Methods Binding and transactivation assays BET bromodomain inhibitor Binding vectors for His-tagged protein production were prepared by inserting the ligand-binding domain cDNA of human LXR�� (amino acids 205�C447) in pET28 (Novagen, Gibbstown, NJ, USA) and the ligand-binding domain cDNA of LXR�� (amino acids 216�C461) in pET24D. Proteins were expressed in Escherichia coli and purified on Ni+ columns. Binding assays using LXR�� and LXR�� protein were run by adding reagents to Wallac Isoplate 1450�C514. Briefly, each 96 plate well contained assay buffer (20 mM Tris pH 7.5, 80 mM NaCl, 2 mM dithiothreitol, 0.125% Chaps and 10% glycerol), 0.1 mg SPA beads (polylysine-coated yttrium silicate beads, RPNQ0010P, GE Healthcare, Piscataway, NJ, USA), LXR�� (0.5 ��g) or LXR�� (0.

25 ��g), 30 nM 3H-ligand (Tularik T0901317, specific activity of 473 Kbq nmol?1) and test compound in a 10-point dose-response dilution. The assay mixture was shaken gently for 2 h on a plate shaker after which the beads were allowed to settle for 1 hour before counting. Transactivation vectors were prepared by inserting the ligand-binding domain cDNA sequences of human or mouse LXR�� and LXR�� in frame with the yeast Gal4 transcription factor DNA binding domain and the nuclear localization signal from the T-antigen of polyoma virus in the eucaryotic expression vector pSG5 (Stratagene, La Jolla, CA, USA). The ligand-binding domain cDNA of human LXR�� and LXR�� was the same as mentioned previously. The mouse sequence corresponded to amino acids 203�C445 for LXR�� and amino acids 201�C446 for LXR��.

The vectors were co-transfected with a pGL3 luciferase reporter plasmid containing a minimal SV40 promoter (Promega, Madison, WI, USA) and five copies of the UAS Gal4 recognition site into U2/OS osteosarcoma cells. Ligands were added as 10-point dose-response curves and then luciferase activity was measured after 48 h. Macrophage RCT All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of the Netherlands Organization for Applied Scientific Research (TNO) or by the Ethics Review Committee on Animal Experiments (Gothenburg region). The animals received food and water ad libitum. Body weight and food intake were monitored during the study. Macrophage RCT was determined essentially as described by Naik et al. (2006).

J774A1 macrophages (obtained from Deutsche Sammlung von Mikroorganismens and Zellkulturen, DSMZ, Braunschweig, Germany) were cultured in RPMI supplemented with 10% fetal bovine serum and radiolabelled with 5 ��Ci mL?1[3H]cholesterol (specific activity 40�C60 Ci/mmol, PerkinElmer, Waltham, MA, USA) together with 100 ��g mL?1 acetylated LDL (Intracel, Rockville, MD, USA) and 1% BSA Dacomitinib (Sigma, St Louis, MO, USA) for 48 h. The J774A1 cells were then washed with phosphate-buffered saline (PBS) and resuspended in medium. The viability was estimated to be ~75% using Trypan blue.

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