Figure 3

Drug-induction of the 264-bp PBRU is abolished by a dominant-negative CXR allele. A, CXR was subcloned into the pHook-2 expression plasmid (Hook-2) either full-length CXR in positive orientation (CXR+), negative orientation (CXR-), lacking its N-terminal amino acids 1–29 (ΔN-term), full-length CXR with four of its cysteine residues (cysteine 31, 34, 83 and 86) in the DNA-binding domain mutated (DBD) or lacking its C-terminal amino acids 383–391 containing the activation function AF-2 (ΔAF2). B, Electrophoretic mobility shift assays with mock in vitro transcribed/translated reticulocyte lysate (lane 1), expression plasmid pHook-2 (lane 2) and either expression plasmids for the different CXR alleles alone (lanes 3–7) or together with a pSG5-expression plasmid for chicken RXRγ (lanes 8–12). The arrow indicates the specific shift of CXR/RXR complexes with the radiolabeled CYP2H1 264-bp PBRU. C, pHook-2 expression plasmids without insert or containing the various CXR alleles were co-transfected with the CYP2H1 264-bp PBRU in the pBLCAT5 reporter vector as well as a lacZ-expression vector for normalization of transfection efficiencies into non-drug responsive CV-1 cells. After transfection, the cells were treated with either vehicle or 400 μM PB for 24 hours before cells were lysed and analysed for reporter gene expression and β-galactosidase expression. Values are the average of the relative CAT expression normalized for β-galactosidase levels of three independent experiments and error bars represent the standard deviation. D, pHook-2 expression plasmids without insert or containing the various CXR alleles were co-transfected with the CYP2H1 264-bp PBRU in the pBLCAT5 reporter vector into drug-responsive LMH cells expressing endogenous CXR. After transfection, the cells were treated with either vehicle or 400 μM PB for 24 hours before cells were lysed and analysed for reporter gene expression and β-galactosidase expression. Values are the average of the relative CAT expression normalized for β-galactosidase levels of three independent experiments and error bars represent the standard deviation.