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. 1998 Feb 1;12(3):331–342. doi: 10.1101/gad.12.3.331

Figure 3.

Figure 3

 In vitro transcriptional activation by ER requires the AF-2 domain and is blocked by anti-estrogens. (A) Transcriptional activation by ER is ERE- and AF-2-dependent. pERE (four EREs) and pIE0 [which is identical to pERE, except that it does not contain any EREs (no EREs)] were assembled into chromatin with ER, ER(L540Q), and/or E2 and then subjected to in vitro transcription–primer extension analysis. The final concentrations of receptor and E2 in the transcription reactions were 4.5 and 30 nm, respectively. (B) ER and ER(L540Q) are not degraded during chromatin assembly. Aliquots of chromatin assembled with the indicated components were subjected to Western blot analysis with anti-ER antibodies. (C) Anti-estrogens inhibit E2-dependent activation by ER with chromatin templates in vitro. Chromatin assembly–in vitro transcription reactions were performed with ER, E2, TOT, and ICI, as indicated. The final concentrations of ER, E2, and anti-estrogens in the transcription reactions were 4.5 nm, 15 nm, and 3 μm, respectively. (D) ER is not degraded during chromatin assembly in the presence of anti-estrogens. Aliquots of chromatin assembled with the indicated components were subjected to Western blot analysis with anti-ER antibodies.