PCR products were electrophoresed on 2% agarose gels. HTC HPLC separation of retinoids and carotenoids from tissues and plasma Retinoids and carotenoids were extracted from tissues and plasma under a dim red safety light (600 nm). Briefly, tissues (20�C40 mg) were homogenized in 200 ��l 2 M hydroxylamine (pH 6.8) and 200 ��l methanol with a glass homogenizer. For determination of ��,��-carotene blood levels, 200 ��l plasma was added to 200 ��l methanol. Then 400 ��l acetone was added to either the plasma or tissue extracts. Extraction of carotenoids and retinoids was performed with petroleum ether. The extraction was repeated 3 times, and the collected organic phases were dried under a stream of nitrogen and dissolved in HPLC solvent.
HPLC separation of carotenoids and retinoids and quantification of peak integrals was performed as described previously (24). Solvents for HPLC and extraction were of HPLC-grade and purchased from Merck (Darmstadt, Germany). RNA preparation and qRT-PCR RNA preparation and qRT-PCR analyses were performed as described previously (25). The following primers were used for qRT-PCR analysis of target genes: ISX, 5��-TTCCACTTCACCCATTACCC-3�� and 5��-CTCTTCTCCTGCTTCCTCCA-3��; SR-BI, 5��-CTCTCCCACCCCCACTTT-3�� and 5��-TTCCCTGTTTGCCCGATG-3��, BCMO1, 5��-ACACCATCCCCGACTTCAC-3�� and 5��-GTTTACCGCCACATACTTCC-3��; and BCMO2, 5��-ATCGCCCAGTTTTGAAGGAG-3�� and 5��-ACCCGAGCAGAGACAGCA-3��. As a housekeeping gene, we used ��-actin: 5��-ACGGGCATTGTGATGGACTC-3�� and 5��-GTGGTGGTGAAGCTGTAGCC-3��.
RNA was extracted from mouse intestine or cultured cells with the Trizol reagent (Invitrogen) and purified by using the RNeasy system (Qiagen). Approximately 2 ��g of total RNA was reverse transcribed with the High Capacity RNA-to-cDNA kit (ABI) following the manufacturer��s instructions. Quantitative PCR (Q-PCR) was carried out using TaqMan chemistry, TaqMan Gene Expression Master Mix and Assays on Demand probes (ABI) for mouse ISX (Mm01243745_m1), mouse Bcmo1 (Mm00502437_m1), mouse Scarb1/SR-BI (Mm00450236_m1), and human ISX (Hs01368145_m1), respectively. The 18s rRNA (4319413E) or ��-actin probe set (ABI) were used as endogenous controls. All real-time experiments were performed with the ABI Step-One Plus qRT-PCR machine. Gene expression analysis was accomplished by the relative standard curve method (ABI Technical Bulletin No. 2).
In silico promoter analysis We used NUBIScan version 2.0 software (http://www. nubiscan.unibas.ch/software) and AliBaba 2.1 (http://www. gene-regulation.com) to identify putative nuclear receptor binding sites in Carfilzomib the human ISX gene. Statistical analysis Student��s t test was used to analyze the data, presented as means �� sd. Values of P �� 0.05 were considered significant. RESULTS ISX expression is under the control of RA and RARs Animal studies indicate that ISX represses intestinal SR-BI and BCMO1 expression (15, 18).