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. Author manuscript; available in PMC: 2011 Nov 16.
Published in final edited form as: Mol Cell. 2009 Sep 24;35(6):755–768. doi: 10.1016/j.molcel.2009.09.015

Figure 3. Independent and cooperative binding of PGC-1α and MED1 to the UCP-1 enhancer dependent upon TRα-RXRα and thyroid hormone.

Figure 3

Standard recruitment assays with proteins and T3 added as indicated were carried out as described in Experimental Procedures and bound proteins were detected by immunoblot.

(A). MED1-independent, T3-dependent binding of PGC-1α to the TRα-RXRα-UCP-1 enhancer complex.

(B). PGC-1α independent binding of MED1, as well as cooperative binding of intact MED1 and PGC-1α, to the TRα-RXRα-UCP-1 enhancer complex.

(C) and (D). Competitive binding of MED1(580–701) and PGC-1α to the TRα-RXRα-UCP-1 enhancer complex. All factors were added concommitantly to the immobilized DNA template. as indicated. In panel D the amount of PGC-1α in lane 4 was 10 times the amount in lane 3 and in the analyses in panel C

(E). Active displacement of prebound PGC-1α from the TRα-RXRα-UCP-1 enhancer complex by MED1(580–701). In lane 2 and 5, “1” and “2” indicate the order of addition of the factors to the preformed TRα-RXRα-UCP-1 enhancer complex. Factor 1 was added first and incubated for 30 minutes prior to addition of factor 2 and incubation for another 30 minutes