E₂-dependent interactions of HeLa nuclear extract proteins with ERα and ERβ LBDs. Immobilized GST (lanes 2 and 8), GST–ERαLBD (lanes 3 and 4) and GST–ERβLBD (lanes 6 and 7) proteins were incubated with HeLa nuclear extract in the absence (−) or presence (+) of 1 μM E₂, and bound proteins were eluted and analyzed by SDS/PAGE and silver staining as described in Materials and Methods. Purified TRAP/Mediator complex from f:Nut2-expressing cells is analyzed in lane 5. Standard molecular mass markers (SM) with sizes in kDa indicated on the left were present in lane 1. Bands marked with an asterisk represent degradation products of GST–ER fusion proteins.

E₂-dependent interactions of TRAP/Mediator with ERα and ERβ LBDs in nuclear extract. Immobilized GST (lane 2), GST–ERαAB (lane 3), GST–ERαLBD (lanes 4 and 5), GST–ERβAB (lane 6), and GST–ERβLBD (lanes 7 and 8) proteins were incubated with HeLa nuclear extract in the absence (−) or presence (+) of 1 μM E₂, and bound proteins were eluted and analyzed by SDS/PAGE and Western blot (with antibodies to proteins indicated on the left) as described in Materials and Methods. A 1/10th equivalent of the input nuclear extract is shown in lane 1. TRAP/Mediator and TR–TRAP complexes immunopurified from cells expressing f:Nut2 and f:TR, respectively, are shown in lanes 9 and 10. The band observed in lane 6 with MED7 antibody is a nonspecific band that crossreacts with this antibody.

TRAP220-dependent interactions of TRAP/Mediator with ER LBDs in nuclear extract. Immobilized GST (lanes 2 and 11), GST–ERα LBD (lanes 3–6), and GST–ERβ LBD (lanes 7–10) were incubated with nuclear extracts from wild-type (WT, lanes 2, 3, 4, 7, and 8) and TRAP220^−/− (KO, lanes 5, 6, 9, 10, and 11) MEFs in the absence (−) or presence (+) of 1 μM E₂, and bound proteins were eluted and analyzed by SDS/PAGE and Western blot (with antibodies to proteins indicated on the left) as detailed in Materials and Methods. One-tenth equivalents of input nuclear extracts were analyzed in lanes 1 (WT) and 12 (KO).

Direct interactions of purified TRAP/Mediator with LBDs of ERα and ERβ. Immobilized GST (lanes 6 and 7), GST–ERα LBD (lanes 2 and 3), and GST–ERβ LBD (lanes 4 and 5) were incubated with immunopurified TRAP/Mediator complex from cells expressing f:CDK8/SRB10 in the absence (−) or presence (+) of 1 μM E₂. After extensive washing, bound proteins were eluted and analyzed by SDS/PAGE and Western blot (with antibodies to proteins indicated on the left) as described in Materials and Methods. A 1/10th equivalent of the input TRAP/Mediator preparation was analyzed in lane 1.

Intracellular association of TRAP/Mediator with ERαΔAB. C8 cells that express FLAG-tagged ERα lacking the AB domain (f:hERαΔAB) were maintained in DMEM-PO₄ medium supplemented with 10% calf serum. f:ERαΔAB and associated proteins were affinity-purified from C8-derived nuclear extract on M2 agarose. Proteins bound in the presence (+) or absence (−) of 1 μM E₂ during purification were eluted with FLAG peptide and analyzed by SDS/PAGE and Western blot with antibodies to proteins indicated on the left. M2 agarose-bound proteins from HeLa extract were analyzed in lane 1, and a 1/10th equivalent of C8 nuclear extract input was analyzed in lane 4.

TRAP/Mediator interactions with full-length ERα and ERβ. (A) Ligand-dependent interactions between TRAP/Mediator and ERs in HeLa nuclear extract. M2 agarose-immobilized FLAG-ERα and FLAG-ERβ were incubated with HeLa nuclear extracts in the absence (−) or presence (+) of 1 μM E₂, and bound proteins were eluted with FLAG peptide and analyzed by SDS/PAGE and Western blot (with antibodies to proteins indicated in the left) as described in Materials and Methods. As a control, HeLa nuclear extract proteins bound to M2 agarose alone were analyzed in lane 2. A 1/10th equivalent of input nuclear extract was analyzed in lane 1. (B) Purified f:TRAP220AB TRAP/Mediator complex. The complex was affinity-purified from cells expressing a FLAG-tagged TRAP220 lacking the C-terminal domain (f:TRAP220AB) and analyzed by SDS/PAGE and silver staining (lane 2). Lane 1 shows HeLa nuclear extract proteins bound nonspecifically to M2 agarose. (C) Direct interactions between purified TRAP/Mediator complex and full-length ERβ. M2 agarose-immobilized TRAP/Mediator (f:TRAP220AB) complex (lanes 3, 4, and 6) and M2 agarose alone (lanes 2 and 5) were incubated with ³⁵S-labeled (in vitro translated) full-length ERβ in the presence (lane 3) or absence (lane 4) of 1 μM E₂ or with control lysate (lanes 5 and 6). After washing, bound proteins were eluted with FLAG peptide and analyzed by autoradiography (Upper) or Western blot with antibodies to the indicated components of TRAP/Mediator complex (Lower). Lane 1 shows 1/20th of the ³⁵S-labeled full-length ERβ input.

TRAP/Mediator complex directly mediates ER function in vitro. (A) Recombinant FLAG-tagged ER proteins. Affinity-purified ERs were analyzed by SDS/PAGE and either Coomassie brilliant blue R-250 staining (lanes 1 and 2) or Western blot with corresponding antibodies (lanes 3 and 4). (B) ER-dependent in vitro transcription. Reactions contained purified general TFs and cofactors, purified TRAP/Mediator complex, and ER proteins (20 nM) as indicated and both 4EREΔ53 G-less reporter and ML200 G-less control templates. TRAP/Mediator was purified from the cell line expressing f:TRAP220AB (Fig. 6B).