FIG. 6.

Role of NR boxes in TRAP220 coactivator function. (A) In the GST pull-down assay, the AB (lanes 1 to 4), a (lanes 5 to 8), b (lanes 9 to 12), and ab (lanes 13 to 16) complexes were incubated with GST (lanes 2, 6, 10, and 14) or with GST-TRα (lanes 3, 4, 7, 8, 11, 12, 15, and 16) either with no ligand (lanes 3, 7, 11, and 15) or in the presence of 10⁻⁷ M T₃ (lanes 2, 4, 6, 8, 10, 12, 14, and 16). Half of the input is shown for each complex tested (lanes 1, 5, 9, and 13). The retained material was eluted and, following SDS-PAGE, was visualized by silver staining. (B to D) In vitro transcription assay mixtures were reconstituted from highly purified factors (16, 22), and transcription of a template bearing TR cognate sites (TRE) was monitored. Purified complexes (derived from AB or mutants a, b, 1, 2, 3, Δ1, Δ2, and ab) were added to the reaction mixtures as indicated. These reaction mixtures also contained TR that was purified via a baculovirus (bv) expression system. bv-expressed RXRα was also included as indicated. The TR-TRAP complex was used as a reference for the experiment shown in panel B; for panels C and D, mutant complexes 2 and 1 also served as references. All reaction mixtures contained RXRα; no ligand was added. Reaction mixtures also contained ML200 template to monitor TR-independent effects, but, at the exposure shown, transcription from this template was not detected. For experiments illustrated in panels C and D, the RNA transcripts were quantitated by phosphorimaging. Transcription levels (rel txn) relative to those obtained in the absence of TRAP/Mediator (lane 2) are indicated. Experiments similar to those shown here were repeated several times. Although the precise magnitude of the effects varied, the general trends as reflected in the data shown were consistently observed. The fold stimulation (and other numbers mentioned in the text) are based on quantitation of the data shown. (E) Transient transfection assays were performed with TRAP220^−/− MEF cells. Plasmid constructs containing either the parental TRAP220 AB fragment or the indicated AB derivative were transfected into the cells in either the presence or absence of T₃. The empty vector was used as a control. Following transfection, the normalized luciferase activity of the resulting extracts was measured. (F) Immunoblotting to monitor expression of the TRAP220 proteins. After TRAP220 and mutant AB proteins were transiently expressed in TRAP220^−/− MEF cells, the expressed nuclear proteins were extracted and probed with anti-FLAG antibodies.