Figure 3.

VDR binding activity in VAT by the EMSA radioactive method. Combined gel shift and supershift experiments were performed with equal amounts of nuclear extracts of VAT from control (lanes 1, 4, and 7), LIR-OM (lanes 2, 5, and 8) and HIR-OM (lanes 3, 6, and 8) to show the ability of VDR to bind to the ³²P-labeled osteopontin VDRE sequence. Nuclear extracts were preincubated with VD as indicated in the panel A. Equal amounts of bacterially expressed wild-type GST (lanes 1, 2, and 3), GST-SRC1 (lanes 4, 5, and 6), and anti-VDR (lanes 7, 8, and 9) were added as indicated. Protein-DNA complexes were resolved from free probe through 8% non-denaturing polyacrylamide gels. Lane 10 of panel B represents the probe alone without nuclear extract, used as a negative control of the system.