Figure 4.

NIX1 associates with DNA-bound nuclear receptors. EMSAs were performed by incubating purified recombinant nuclear receptor proteins with their ³²P-labeled cognate response elements. DNA-protein complexes were subsequently challenged with recombinant GST alone or GST-NIX1 (see Materials and Methods for details). (A) His-tagged RORβ was incubated with the ³²P-labeled palindromic P0 response element. Addition of recombinant GST-NIX1 (2 μg) in contrast to GST alone results in a clear supershift indicating ternary complex formation of DNA-bound RORβ and NIX1. GST-NIX1 by itself does not bind to the response element P0. (B) Purified recombinant GST-RARα and GST-RXRα were bound to the ³²P-labeled cognate DR5 response element. Resulting heterodimeric receptor-DNA complexes are specifically retarded by GST-NIX1 only in the presence of all-trans-retinoic acid (1 μM), but not in the absence of ligand. GST-NIX1 does not bind the response element.