For the cycloheximide experiment, cells were pretreated with 20 ��g/ml of cycloheximide for 20 min, and RA (1 ��M) was added and cells were incubated for another 4�C6 h at 37��C in 5% CO2. For Nutlin-3a buy the RAR pan-antagonist LE540 experiment, CaCo-2 cells were pretreated with 1 ��M of LE540 for 1 h; after which 1 ��M RA was added, and cells were incubated for an additional 4 h. Control cells were not treated or were treated with 1 ��M RA alone for 4 h. After harvesting, cells were then processed for total RNA. mRNA expression of relevant genes was determined by qRT-PCR with gene-specific probe sets (ABI). HepG2 cells were cultured in 100-cm2 dishes and transfected with 4�C6 ��g of purified plasmid DNA (pISX-WT) by using LipofectAMINE 2000 (L2000) and Opti-MEM according to manufacturer��s instructions (Invitrogen).

Cells were harvested 48�C72 h post-transfection, and total protein was extracted and processed as detailed below. Protein isolation and Western blot analyses Total protein from animal tissue or cultured cells was isolated at indicated time points by using the M-PER mammalian protein extraction reagent with protease inhibitors (Roche, Basel, Switzerland) according to manufacturer��s instructions (Pierce). Mice were sacrificed by cervical dislocation. Small intestines were collected, rinsed in ice-cold phosphate buffered saline (PBS; pH 7.4), and snap-frozen in liquid nitrogen. Mouse intestine (~100 mg) was then homogenized in liquid nitrogen using a mortar and pestle and transferred directly into M-PER buffer (500 ��l) containing protease inhibitors.

Proteins (30�C50 ��g) were fractionated on 4�C10% SDS-PAGE gels using the Bio-Rad Minigel system and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Equal protein loading was confirmed by routine immunoblotting of the membranes with Ponceau S staining and by Western blot analysis using anti-RAN as the loading control. PVDF membranes were blocked with 5% milk prepared in Tris-buffered saline (pH 7.4) containing 0.05% Tween (TBS-T) for 1 h and then probed with either anti-ISX, anti-SR-BI, or anti-BCMO1 (1:1000 dilution) antibody overnight at 4��C, followed by incubation with the appropriate HRP-conjugated secondary antibody, before being visualized with the enhanced ECL chemiluminescence detection system (Pierce or Pharmacia). For determination of liver retinol binding protein (RBP4) levels, Anacetrapib a commercially available polyclonal antiserum raised against human RBP4 (DakoCytomation, Hamburg, Germany) was used in a 1:1000 dilution. WT ISX plasmid construction Total RNA (1 ��g) from the human colonic CaCo-2 cell line was reverse transcribed by using the SuperScript One-Step RT-PCR for LongTemplates system (Invitrogen).