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Mammalian two-hybrid assay

WL Wenwei Lin
YW Yue-Ming Wang
SC Sergio C. Chai
LL Lili Lv
JZ Jie Zheng
JW Jing Wu
QZ Qijun Zhang
YW Yong-Dong Wang
PG Patrick R. Griffin
TC Taosheng Chen
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The mammalian two-hybrid assay method has been described previously35. Briefly, the CheckMate mammalian two-hybrid system (Promega) was used to evaluate the association between hPXR and its co-regulators. LS180 cells were co-transfected with an hPXR plasmid (pACT-hPXR, pACT-hPXRM429A or pACT-hPXRF429A), a coregulator plasmid (pBIND-SRC-1, pBIND-TIF2, pBIND-SMRTτ [amino acids 2077–2471], or pBIND-mNCoR [amino acids 1958–2401]), and a GAL4 firefly luciferase reporter plasmid (pG5-luc). The Renilla luciferase is constitutively expressed from the pBIND vector, and served as an internal transfection control. The activity of the firefly and Renilla luciferase was measured by using the Dual-Glo Luciferase Assay (Promega), and the relative luciferase activity of pG5-luc was determined by normalizing the activity of firefly luciferase to that of Renilla luciferase.

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