Figure 2
From: Isolation of novel coregulatory protein networks associated with DNA-bound estrogen receptor alpha
Large-scale agarose gel electrophoresis and complex analysis. A. Large-scale reactions containing unlabeled ERE-containing oligos were incubated with HeLa nuclear extracts and an ERα-specific antibody in the absence (lane 2) or presence (lane 3) of purified ERα. Small-scale reactions containing 32P-labeled ERE-containing oligos, HeLa nuclear extracts, an ERα-specific antibody, and purified ERα were also prepared and run in parallel to indicate the location of the protein-DNA complexes (*, lanes 1 and 4). E2 was included in all binding reactions. Complexes were resolved on an agarose gel and were visualized in the wet gel by autoradiography. Gel regions comigrating with the 32P-labeled protein-ERα-ERE complexes were excised (boxed areas) and contained unlabeled DNA and associated proteins without (lane 2) or with (lane 3) ERα and ERα-specific antibody. B. Proteins were isolated from the excised agarose gel pieces and subjected to Western blot analysis with antibodies directed against ERα, AIB-1, p300, or Pol II.