Thus, the unique feature of hCAR1+A may eventually provide a new system that could facilitate our efforts in delineating the mechanisms of hCAR activation. Materials and Methods Chemicals and Biological Reagents. PB, phenytoin (PHN), rifampicin (RIF), artemisinin (ART), carbamezapine selleck kinase inhibitor (CMZ), WY-14643, 1,4-bis[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP), clotrimazole (CLZ), butylated hydroxyanisole (BHA), PK11195, chenodeoxycholic acid (CDCA), 22(R)-hydroxycholesterol (HOC), diazepam (DAP), methadone (MD), 3-methylcholanthrene (3MC), and meclizine (MLZ) were purchased from Sigma-Aldrich (St. Louis, MO). Okadaic acid (OA) was purchased from Calbiochem (San Diego, CA). CITCO was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA).

Efavirenz (EFV) was purchased from Toronto Research Chemicals (Toronto, ON, Canada), and nevirapine (NVP) was purchased from U.S. Pharmacopeia (Rockville, MD). Fluconazole (FLU) and myclobutanil (MCB) were purchased from LKT Laboratories (St. Paul, MN). The Dual-Luciferase Reporter Assay System was purchased through Promega (Madison, WI). FuGENE 6 and Fugene HD transfection reagents were obtained from Roche (Basel, Switzerland). Other cell culture reagents were purchased from Invitrogen (Carlsbad, CA) or Sigma-Aldrich. Plasmids Constructions. The pCR3-hCAR1, pEYFP-hCAR1, GST-hCAR1, pcDNA3.1-mSRC-1, and pcDNA3.1-GRIP-1 were provided by Dr. Masahiko Negishi (National Institute of Environmental and Health Sciences, National Institutes of Health, Research Triangle Park, NC). The pCMV2-hCAR3 expression vector and CYP3A4-PXRE/XREM luciferase reporter construct were obtained from Drs.

Curtis Omiecinski (Pennsylvania State University, University Park, PA) and Bryan Goodwin (GlaxoSmithKline, Research Triangle Park, NC), respectively. The CYP2B6-PBREM/XREM reporter was described previously (Wang Dacomitinib et al., 2003). Plasmids used for mammalian two-hybrid assay, pG5-Luc and pACT, were obtained from Promega. pACT-hCAR1, pM-SRC-1 (621�C765), and pM-GRIP-1 were generated as described previously (Ueda et al., 2005). The pEYFP-hCAR3, GST-hCAR3, and pACT-hCAR3, vectors were constructed by subcloning the full-length hCAR3 into the multicloning sites of pEYFP-c1, pGEX4T-3, or pACT, respectively. The pRL-TK Renilla luciferase plasmid used to normalize firefly luciferase activities from Promega. Generation of hCAR Chimeric Constructs. Human CAR chimeras (Fig. 1A) were generated by introducing appropriate nucleotides corresponding to the residues of the five-amino-acid insertion of hCAR3 into the pCR3-hCAR1 expression vector by use of the QuikChange Site-directed Mutagenesis System (Stratagene, La Jolla, CA). The mutagenic primers used for constructing the chimeras were summarized in Table 1.