FIGURE 3.

Mediator containing FLAG-MED25-VWA is not efficiently recruited to promoter DNA in vitro by ATF6α. Promoter binding assays were performed using Mediator purified through FLAG-MED25 or FLAG-MED25-VWA, nuclear extracts that had been depleted of Mediator, and either the GAL4 DNA binding domain (GAL4) or a GAL4-ATF6α-AD fusion protein containing ATF6α amino acids 1–150. A, Western blots of representative Mediator subunits present in 3% (lanes 1, 4, and 7), 9% (lanes 2, 5, and 8), or 27% (lanes 3, 6, and 9) of input binding reactions prior to separation of DNA-bound and unbound fractions. B, Western blots of representative Mediator subunits in DNA-bound fractions. Proteins were detected using the indicated antibodies.