FIG. 1.
Direct binding of CAR to FOXO1. (a) Mammalian two-hybrid assay. The pG5-Luc reporter plasmid was cotransfected with various combinations of pBIND, pACT, pBIND/mCAR, and pACT/mFOXO1Ct into HepG2 cells. The amount of each plasmid was 0.2 μg, and the total amount of plasmids was adjusted by pcDNA3-V5-His. At 24 h after transfection, cells were treated with 0.1% DMSO, TCPOBOP (250 nM), or androstenol (10 μM) for an additional 24 h. Subsequently, cells were harvested and cell extracts were prepared for dual-luciferase assays. Relative activities were calculated by taking the activity obtained from the GAL4DBD- and VP16AD-transfected cells in the presence of DMSO as one. Bars indicate means ± standard deviation. (b) GST pull-down assay. In vitro-translated 35S-labeled FOXO1 and CAR were incubated with bacterially expressed GST-mFOXO1 and GST-mCAR fusion proteins, respectively, in the presence of 0.1% DMSO, 1 μM TCPOBOP (T), 10 μM androstenol (A), or both (T/A). GST was used as a negative control for binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography were done as described in Materials and Methods.