Luminol detection kit

Whole-cell lysates were prepared as described previously [4 ], by lysing the cells in boiling buffer (1% SDS, 1 mM sodium vanadate, 10 mM Tris [pH 7.4]) in the presence of complete protease inhibitor mixture (Roche diagnostics). The viscosity of the samples was reduced by sonication. Whole-cell lysates or immunoprecipitation samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and electroblotted onto a nitrocellulose membrane (GE Healthcare). After incubation for one hour at RT with 5% nonfat milk in Tris-buffered saline (TBS)–0.1% Tween-20, membranes were incubated overnight with the primary antibody diluted in TBS-milk-Tween, washed, incubated with the secondary antibody for 30 minutes at RT, and washed again before analysis with a luminol detection kit (Santa Cruz biotechnology) and chemidoc analyser (Bio-Rad, Marnes-la-coquette, France). Relative quantification was performed using ImageLab software. The following mouse mAbs were used: anti–β-actin (A1978) from Sigma-Aldrich, anti- LXRβ (PP-K8917) from R&D sytems. We also used rabbit pAbs anti-RXRα (ΔN197 - Santa Cruz biotechnology) and anti-GFP (PA1-980A – Fisher scientific, Illkirch, France). Secondary Abs HRP-conjugated polyclonal goat anti-mouse and swine anti-rabbit immunoglobulins (Jackson ImmunoResearch, Suffolk, UK) were also used.

Immunoblot analysis was performed for different cell phenotypes by the method described previously^{67 (link)}. Total cellular proteins (50 μg/lane) were separated on 12% polyacrylamide gel and electro-transferred on PVDF membranes (Millipore Corp, USA). The membrane was blocked in PBS containing 5% non-fat skimmed milk and probed with specific antibodies against cFos (H-125), Fra-1 (R-20), cJun (N), JunB (210) and JunD (329) by incubating the membrane overnight in pre-standardized dilution of primary antibody in blocking solution at 4 °C. These blots were washed, incubated with HRP-anti-mouse IgG secondary antibodies and visualized by Luminol detection kit (Santa Cruz Biotech) by exposing the blot to KODAK X-Omat films (Kodak India, India). The Western blot membranes were stripped and reprobed for β-actin (C-11) expression which was used as an internal control. The quantitative densitometric analysis of the bands was performed using Alpha Ease FC version 4.1.0 (Alpha Innotech Corporation, IL).