FIG. 2.

Binding of NRs to Fli-I in vitro and in vivo. (A) In vitro binding with ER. LRR and gelsolin-like fragments of Fli-I were translated in vitro and were tested for binding to bead-bound GST or GST-ER in the absence or presence of E2. (B) Coimmunoprecipitation with ER. Flag-tagged LRR or gelsolin-like fragments of Fli-I (2.5 μg of plasmid) were coexpressed with ER (2.5 μg of plasmid) in Cos-7 cells in the presence or absence of E2. Immunoprecipitation (IP) by anti-Flag antibody was followed by immunoblotting (W) with anti-ER antibody (upper panel). Expression of ER in the transfected-cell extracts before immunoprecipitation (input) was examined by immunoblotting with anti-ER antibodies (lower panel). (C) Coimmunoprecipitation with TR and CARM1. Coimmunoprecipitation of the Fli-I fragments with TR (2.5 μg of plasmid) or HA-tagged CARM1 (2.5 μg of plasmid) was tested as done for panel B in the presence and absence of T3, except that anti-TR (lanes 1 to 6) or anti-HA (lanes 7 and 8) was used for immunoprecipitation, and Fli-I fragments were detected on the subsequent immunoblot (W) with anti-Flag antibodies. Expression of LRR and gelsolin-like fragments of Fli-I in the transfected-cell extracts was examined before immunoprecipitation (input) by immunoblot with anti-Flag antibodies (lower panel).