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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: Nat Struct Mol Biol. 2014 Nov 10;21(12):1028–1034. doi: 10.1038/nsmb.2914

Fig. 4. MED14-dependent Mediator-Pol II interaction and MED26-dependent Pol II recruitment in nuclear extract.

Fig. 4

(a) SDS-PAGE analysis (silver staining) of purified preparations of Pol II, TFIID and natural Mediator used in the binding assays. (b) Western blot analysis of Mediator interaction assays. Binding reactions included the Mediator head module and Pol II or TFIID, as indicated. Anti-MED30 immunoprecipitates were probed for TFIID (TBP), Pol II (RBP1), and selected Mediator subunits. (c) Western blot analysis of Mediator head interaction assays in the presence of TFIIF. Binding reactions were as in panel b, except that they also included TFIIF as indicated. (d) Western blot analysis of Mediator-Pol II interaction assays. Binding reactions included Pol II and the indicated recombinant Mediator sub-complexes (H, H+M, or H+M+14). Anti-MED30 immunoprecipitates were probed for Pol II (RPB1 and RBP6) and Mediator (MED7, MED14, MED30) subunits. For lanes 1-3 (inputs), longer Western blot exposures are also included. (e) Western blot analysis of an immobilized template recruitment assay to assess Pol II recruitment. Reactions were done with control or Mediator-depleted Hela NE (ΔMediator NE) and were supplemented with various recombinant Mediator sub-complexes. Recruitment of Pol II (RPB1) and TFIID (TBP) was monitored. See also Supplementary Data Set 2.