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. 1999 Mar 2;96(5):1959–1964. doi: 10.1073/pnas.96.5.1959

Figure 3.

Figure 3

Absence of CBP/p300 and SRC-1/TIF-2 in the immunopurified f:TRα/TRAP complex and the in vitro assay system. (A) The f:TRα/TRAP complex. Immunopurified f:TRα/TRAP complex (40 ng of f:TRα, 266 ng of total protein including TRAPs) and HeLa nuclear extract (30 μg) were assayed by Western blot analysis with antibodies to TIF2, SRC-1, CBP, and p300. Only the relevant regions of the blot are shown. (B) Transcription assay components. Purified factors TFIIA (500 ng), f:TFIID (100 ng), TFIIE/F/H (900 ng), RNA polymerase II (600 ng), and USA (250 and 500 ng), as well as nuclear extract (30 μg), were assayed by Western blot analysis with antibodies to p300 and CBP. Only the relevant regions of the blot are shown. The amount of each factor loaded on the gel corresponds to 2–4 times the amount used for a single in vitro transcription reaction (Fig. 1). The general factors TFIIB and TBP were not included in this assay as they were expressed in E. coli and, thus, are devoid of CBP/p300. (C) CBP standard. The indicated amounts (in fmoles) of a recombinant glutathione S-transferase-CBP fusion protein (containing CBP residues 1990–2441) were determined by Western blot analysis with anti-CBP antibodies under the same conditions employed in A and B.