FIG. 2.

Functional analysis of the residual TRAP/Mediator complex from TRAP220^−/− MEFs. (A) Wild-type (wt) TRAP/Mediator complex (f:NUT2, HeLa) and a complex obtained from TRAP220^−/− MEFs were incubated with GST (lanes 2 and 7), GST-VP16 (lanes 3 and 8), or GST-TRα (lanes 4, 5, 9, and 10). In the case of interactions with GST-TRα, incubations were done in either the absence (lanes 4 and 9) or the presence (lanes 5 and 10) of T₃. After being washed, the bound material was eluted from the beads and analyzed by SDS-PAGE and immunoblotting with the indicated TRAP/Mediator antibodies. (B) In vitro transcription reaction mixtures were reconstituted from highly purified transcription factors. All reaction mixtures contained 50 ng of GAL-VP16. TRAP/Mediator was added as indicated: lane 2, TRAP/MED Trap220^−/− (f:NUT2); lane 3, TRAP/MED wt (f:NUT2). (C) In vitro transcription reactions were carried out as described for panel B, except that baculovirus-expressed TRα (10 ng) and RXRα (20 ng) were added to lanes 4 to 6. No exogenous ligand was added.