FIG. 14.

hNRC associates with CBP in vivo, and EIA abrogates both the intrinsic activity and ligand-dependent activation function of hNRC. (A) pEBG vectors expressing GST, a GST fusion of hNRC, or GST-hNRC(1-852) were transfected into 293T cells. Extracts were prepared as described in Materials and Methods and incubated with glutathione-agarose. After washing, the proteins bound to the glutathione-agarose beads were resolved by SDS gel electrophoresis and analyzed for CBP by Western blotting using a polyclonal anti-CBP antibody. Lane 1, a sample of the total lysate prior to incubation with glutathione-agarose beads; lane 4, amount of CBP retained by GST-hNRC; lane 3, GST alone, which does not bind CBP; lane 2, hNRC(1-852), which also does not bind CBP. (B) pBL-G5-CAT2 was cotransfected with expression vectors for the Gal4 DBD or for Gal4-hNRC with or without a vector expressing adenovirus 12S E1A. The cells were incubated with or without 500 nM 9-cis-RA. All samples were analyzed in duplicate, and the experiment was repeated two times with similar results.