FIG. 5.

MED1 phosphorylation promotes its association with the Mediator complex. (A) Isolation of native Mediator complexes containing phosphorylated MED1 and RNA Pol II. Nuclear extract prepared from HeLa cells cultured in FBS was incubated with anti-MED1 or preimmune antibodies. The bound complexes were then eluted and probed by immunoblotting with the antibodies indicated on the right of the panels. HeLa nuclear extract (NE) (10 μg) was loaded as a control. IP, immunoprecipitation. (B to E) HeLa, MCF-7, and HeLa-AR cells were serum starved and then stimulated with (+) or without (−) EGF, E2, DHT, or U0126, as indicated. Nuclear extracts were then prepared and incubated with anti-MED1 antibodies. The bound complexes were first normalized for equivalent MED1 concentrations via anti-MED1 immunoblotting. The normalized blots were then stripped and reprobed for RNA Pol II and other Mediator subunits, as indicated. For panels A to D, the immunoblot signal generated by the anti-phosphothreonine antibody precisely overlapped the band generated by the anti-MED1 antibody on the same blot.