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. 2007 Jan 8;27(5):1844–1858. doi: 10.1128/MCB.01363-06

FIG. 7.

FIG. 7.

Requirement of the mediator for transcription from the X gene promoter. (A) HeLa cell nuclear extract was immunodepleted with anti-MED26 or control (preimmune) antibody. The levels of MED26 protein in the depleted extracts were assayed by immunoblotting with anti-MED26 antibody. The immunodepleted nuclear extracts were then tested for transcriptional activity of the X gene (using the enhancer-X promoter construct pBS-HBXB or using the minimal promoter construct pXMP2CAT) or for transcriptional activity of a U6 snRNA gene that is not regulated by the mediator (control). MED26 depletion specifically abolished transcription of the X gene. (B) In vitro reconstitution confirms that the mediator complex is required for transcription from the X gene. In vitro transcription of the X gene (enhancer-X promoter or X minimal promoter 1), XCPE1 alone, and the Sp1-TATA templates was performed using control (lane 1) or MED26-depleted (lanes 2 to 4) HeLa nuclear extracts (NE) in the absence (lane 2) or presence (lanes 3 and 4) of the mediator (MED26-containing) complexes purified from P.5 (lane 3) or P1.0 (lane 4) phosphocellulose fractions.