Rabbit anti

Synthetic LXR ligand 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl) amino] propyloxy] phenylacetic acid hydrochloride (GW3965) was kindly donated by Jon Collins (GlaxoSmithKline, Research Triangle Park, NC). Dihydroethidium (DHE) and TRIzol Reagent were from Life Technologies (Carlsbad, CA). Mouse monoclonal antibody against LXRα (ab41902) and rabbit polyclonal antibody against LXRβ (ab28479) were from Abcam (Cambridge, UK); rabbit anti-mouse nitrotyrosine antibody (06-284) was from Millipore (Billerica, MA); rabbit anti-cleaved caspase-3 (5A1E, #9664), rabbit anti-nuclear factor kappa-light-chain-enhancer of activated B cell p65 (NF-κB p65, C22B4; #4764), rabbit anti-Akt (#9272), rabbit anti-phospho-Akt (D9E, Ser473, #4060), rabbit anti-p38 mitogen-activated protein kinase (p38 MAPK, #9212), rabbit anti-phospho-p38 MAPK (D3F9, Thr180/Tyr182, #4511), rabbit anti-c-Jun N-terminal kinase (JNK, 56G8, #9258), rabbit anti-phospho-JNK (81E11, Thr183/Tyr185, #4668), rabbit anti-Histone H3 (#9715) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 14C10, #2118) were from Cell Signaling Technology (Beverly, MA). IRDye 800CW goat anti-mouse (926-32210) and anti-rabbit IgG (926-32211) secondary antibodies were from LI-COR Biosciences (Lincoln, NE).

Samples were diluted to a final protein concentration of 2 µg/µl in Laemmli buffer, and analyzed by SDS-PAGE on 10% resolving acrylamide gels. After transferring onto Odyssey nitrocellulose membranes (LI-COR), protein samples on membranes were blocked in 5% (w/v) non-fat milk in Tris-buffered saline solution containing 0.5% Tween-20 (TBST), pH 8.0, and exposed to primary antibodies [Rabbit anti-oestrogen receptor α (ERα) (C1355) polyclonal antibody, 1∶4,000, Millipore, Billerica, MA; Code No. 06-935; Lot No. NG1838275; Rabbit anti-oestrogen receptor β (ERβ) monoclonal antibody, 1∶4,000, Millipore; Code No. 05-824; Lot No. JBC1850147; Rabbit anti-androgen receptor (AR) PG-21 polyclonal antibody, 1∶4,000, Millipore; Code No. 06-680; Lot No. DAM1661059 (Table 4)] diluted in blocking solution, at 4°C overnight. Hybridized membranes then were washed 3 times in TBST for 5 mins, and probed with Odyssey secondary antibodies, IRDye 800CW Goat anti-Rabbit IgG and IRDye 680RD Goat anti-Mouse IgG (LI-COR) (1∶10,000). Immunoreactivity was visualised and analysed on the Odyssey Classic infrared imaging system (LI-COR; Fig. 15).