Sueyoshi, T., Moore, R., Sugatani, J., Matsumura, Y. and Negishi, M.
Notes: This article examines the interaction between CAR, a member of the nuclear steroid/thyroid hormone receptor family, which translocates from the cytoplasm to the nucleus when cells are exposed to phenobaritol, and the membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A), which is a novel CAR-binding protein. The R16A protein, it was expressed using the TnT® Coupled Reticulocyte Lysate System and labeled with 35S. This protein was incubated with GST-hCAR-fusion protein attached to a glutathione resin; the resin was washed, and the bound protein was separated by PAGE and detected by autoradiography. Affinity-tagged R16A was expressed in HepG2 cells, purified and separated by SDS-PAGE. The two major bands were excised, digested with Sequencing Grade Modified Trypsin, lyophilized, resuspended in acetonitrile and subjected to mass spectrometric analyses. The CheckMate™ Mammalian Two-Hybrid System was used to examine the interaction between wildtype and mutated R16A. Forty-eight hours posttransfection into HepG2 cells or 16 hours after DNA injection into mice and liver homogenization, the luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (3752)