Flag epitope

Procedures for lysing yeast cells and immunoblotting have been reported previously.^{33 (link)} Briefly, yeast cells expressing wt Tsa1 or decamer interface variants were grown to mid log phase and lysed with acid-washed glass beads under nondenaturing conditions in lysis buffer (20 mM Tris (pH 8.0), 0.5 mM EDTA, 10% glycerol, 50 mM NaCl, and Protease Arrest protease inhibitor cocktail (G-Biosciences)). Immunoblots were conducted using reducing SDS-PAGE prior to transfer onto PVDF and detection with antibodies against the FLAG epitope (Sigma), TAP tag (antiprotein A, Sigma), or Pgk1 (Invitrogen). Where indicated, quantification of protein expression was performed on digital images of immunoblots using Fiji software, normalizing to Pgk1 expression as a loading control.^{34 (link)} For immunoprecipitation of Tsa1 with cross-linked binding partners, cells were grown to mid log phase and treated for 1 h at 30 °C with the cross-linker divinyl sulfone (DVSF, 1 mM). Subsequently, immunoprecipitation of Tsa1 from yeast lysates was performed with anti-FLAG beads as previously described.^{31 (link),32 (link)} Immunoprecipitates were probed for cross-links between TAP-tagged Trx2 and FLAG-tagged Tsa1 variants using immunoblot.

The pTracer vectors, CMV2-hCAR1, CMV2-hCAR2, CMV2-hCAR3, CMV2-mCAR and CMV2-hPXR (human pregnane X receptor), as well as p3 × FLAG-hCAR1, pcDNA3.1-RXRα, CYP2B6-PBREM (phenobarbital-responsive enhancer module)/XREM (xenobiotic-responsive enhancer module) or CYP3A4-XREM reporter and pRL-TK were used as described previously [27 (link),28 (link)]. The pcDNA3.1-RXRα LBD, VP16 vectors expressing hCAR1-LBD, pM vector expressing GAL4-SRC1 (steroid receptor co-activator)-RID (receptor-interaction domain) and GAL4-GRIP1 (glutamate-receptor-interacting protein 1)-RID used in mammalian two-hybrid experiments were also reported previously [29 (link)]. The p3 × FLAG-ubiquitin plasmid, which expresses the 76-amino-acid ubiquitin protein tagged with three copies of the FLAG epitope (Sigma-Aldrich), was provided by Dr Adriano Marchese (Department of Molecular Pharmacology and Therapeutics, Loyola University, Stritch School of Medicine, Chicago, IL, U.S.A.) and Dr Jeffrey Benovic (Department of Biochemistry & Molecular Biology, Thomas Jefferson University, Philadelphia, PA, U.S.A.). The adenoviral-YFP-fused hCAR vector was provided by Dr Hongbing Wang (Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, U.S.A.). The pCMV6-SUG1 (suppressor for Gal1) was purchased from Origene.