Fig. 2.

Nuclear extracts from CAR3-transfected cells do not bind a DR-4 DNA probe, and CAR3 does not interact with RXR in mammalian 2-hybrid assays. A, Western blot of 40 μg of crude nuclear extract from 24-h transfected COS-1 cells. Nuclear extracts were generated as described under Materials and Methods. Blots were probed with HRP-conjugated M2 FLAG antibody. B, 6 μg of nuclear extract from A was used in an EMSA with a DNA probe representing one copy of the DR-4 element engineered into the DR-4 × 3 reporter. Details of the experiments are found under Materials and Methods. Protein/probe mixtures were incubated with either DMSO or 5 μM CITCO. C, mammalian two-hybrid assays were performed as illustrated in the figure and as described under Materials and Methods. Data are presented as normalized and adjusted values where the luciferase activity of the GAL4/VP16 (empty vector/empty vector) experiment is set to 1. Each data point represents the mean ± S.E. of four separate transfections. *, probe retained in well.