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. 2013 Mar 22;288(19):13655–13668. doi: 10.1074/jbc.M113.455485

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — Ketoconazole disrupts wild-type but not mutant PXR association with coactivator, SRC-1. A, erg3Δ/erg11Δ strain was transformed with the indicated plasmids, and plated colonies were subjected to X-gal lift assay (left panel) and β-gal liquid assay (right panel). Lane 1, pSH empty vector + pGADNOT empty vector; lane 2, pSH-PXR + pGADNOT empty vector; lane 3, pSH empty vector + pGADNOT-SRC-1; lane 4, pSH-PXR + pGADNOT-SRC-1; lane 5, pSH-INI-1 + pGADNOT-c-Myc (positive control). B, shown is a erg3Δ/erg11Δ colony replica in plates containing vehicle (0.2% DMSO; lane 1) or ketoconazole (25 μm; lane 2). An X-gal lift assay (left panel) and β-gal liquid assay (right panel) were then performed. C, erg3Δ/erg11Δ colony replica in plates containing vehicle (lane 1 and 3) or ketoconazole (25 μm; lanes 2 and 4) is shown. T248E/K277Q indicates specific PXR mutant. D, shown are immunoblots of specific proteins as indicated from yeast colonies (lanes 1 and 2, pSH-PXR + pGADNOT-SRC-1; lanes 3 and 4,pSH-PXR T248E/K277Q mutant + pGADNOT-SRC-1) randomly picked from plates containing vehicle (lanes 1 and 3) or ketoconazole (lanes 2 and 4). E, erg3Δ/erg11Δ strain was transformed with the indicated plasmids, and plated colonies were subjected to an X-gal lift assay (left panel) and β-gal liquid assay (right panel). Lane 1, pSH empty vector + pGADNOT empty vector; lane 2, pSH-SRC-1 + pGADNOT empty vector; lane 3, pSH empty vector + pGADNOT-PXR; lane 4, pSH-SRC-1 + pGADNOT-PXR; lane 5, pSH-INI-1 + pGADNOT-c-Myc (positive control). F, procedures were as in B and C. erg3Δ/erg11Δ colony replica in plates containing vehicle (lane 1) or ketoconazole (25 μm; lane 2) are shown. X-gal lift assay (left panel) and β-gal liquid assay (right panel) were then performed.