FIG. 9.
C termini of RXRα and Nur77 are not required for RXRα/Nur77 interaction in solution. (A and B) GST pull-down assays for determination of RXRα/Nur77 heterodimerization. GST-Nur77, GST-RXRα, or GST control protein immobilized on glutathione-Sepharose (20 μl) was incubated with in vitro synthesized 35S-labeled Nur77, RXRα or their mutants (5 μl) as indicated. Bound proteins were analyzed by SDS-PAGE and autoradiography. One of three similar experiments is shown. (C) Coimmunoprecipitation assay for Nur77 and RXRα/385 interaction. Expression vectors for Flag epitope-tagged-Nur77 (Flag-Nur77) and GFP-RXRα/385 were cotransfected into HEK293T cells. The expressed Flag-Nur77 and GFP-RXRα/385 were then immunoprecipitated by using either anti-Flag antibody or control IgG, and immunoprecipitates were examined by Western blotting with anti-GFP antibody. The same membranes were also blotted with anti-Flag antibody to determine precipitation specificity and efficiency. Input represents 5% of total cell extract used in the precipitation assays. One of two similar experiments is shown. (D) Reportergene assay. (NurRE)2-tk-chloramphenicol acetyltransferase (CAT) (100 ng), β-galactosidase (β-Gal; 100 ng), and Nur77 (25 ng) expression vectors were transiently transfected into HEK293T cells together with or without RXRα or RXRα/385. Cells were treated with or without 9-cis-RA (10−7 M) as indicated. CAT activity was determined and normalized relative to β-Gal activity. Bars represent averages ± deviations from three different experiments. (E) Schematic representations of RXRα and Nur77 mutants. The DBD, LBD, and A to F domains are indicated. (F) GST pull-down assays for determination of RXRα/Nur77 heterodimerization. The indicated GST-RXRα, its mutants, or GST control protein was immobilized on glutathione-Sepharose (20 μl) and incubated with in vitro-synthesized 35S-labeled Nur77 or its mutants (5 μl) as indicated. Bound proteins were analyzed by SDS-PAGE and autoradiography. One of three similar experiments is shown.