Fig. 5.

Validation of a panel of PXR-associated coregulatory proteins in the mammalian two-hybrid system. (A) The NRID derived from known PXR-associated coregulatory proteins, including its obligate dimeric partner RXRα; coactivator proteins SRC1, GRIP1, and PBP; and corepressor proteins SMRT and NCoR, were fused to the GAL4 DBD. (B) Expression levels of the GAL4-cofactor NRID were confirmed using anti-GAL4 antibody in Western blot analysis. (C) Expression levels of the wild-type and mutant PXR VP16-fusion proteins were confirmed using anti-PXR antibodies in Western blot analysis. All constructs described here were analyzed for their open-reading frame integrity using DNA sequence analysis, respectively. (D) The interaction between wild-type VP16-PXR and GAL4 cofactors was analyzed. Forty-eight hours post-transfection, reporter gene activity was determined in the presence and absence (0.1% DMSO) of 10 μM Rif for an additional 24 hours. The blank (−) represents CV-1 cells transfected with luciferase and β-gal reporter genes only treated with vehicle or Rif as indicated. WB- Western Blot, UAS)₅- Upstream Activating Sequence. Data are normalized to β-galactosidase and are reported as the avg. ± S.D. (n = 4), in which P < 0.05 (*), P < 0.01 (**), and P < 0.001 (****).