FIG. 2.

TFIIB and 13S E1A affect the binding of cyclin E-Cdk2 complexes to p300, while 12S E1A affects the binding of TFIIB to p300. (A) TFIIB inhibits interactions between cyclin E-Cdk2 and p300. Products of immunoprecipitations performed with antibodies to Cdk2 were incubated with increasing amounts of purified GST (lanes 3 to 5) or GST-TFIIB (lanes 6 to 8), as indicated, followed by the addition of an in vitro-translated carboxy-terminal p300 fragment spanning amino acids 1240 to 1906 [p300(C)]. Complexes were analyzed by SDS–8% PAGE. Lane 1 contains 10% of the in vitro-translated p300 sample, and lane 2 shows the control immunoprecipitate which contains no competitor GST protein. (B) 12S E1A weakly inhibits the interaction of p300 with TFIIB but has no effect on the interaction of p300 with cyclin E-Cdk2. (Left panel) Immunoprecipitations were performed with control antibodies (lane 10) or antibodies to Cdk2 (lanes 11 to 13). GST-12S E1A (lane 13) or an amount of GST exceeding that of GST-12S E1A in lane 13 (lane 12) were added to immunoprecipitates prior to incubation with in vitro-translated COOH-terminal p300 (amino acids 1240 to 2412) (similar results were obtained with p300 [amino acids 1240 to 1906; data not shown]). The control immunoprecipitate that lacks GST or GST-12S E1A is shown in lane 11. Lane 9 contains 10% of the p300 in vitro translation product. (Right panel) Immunoprecipitations were performed with control antibodies (lane 15) or antibodies to TFIIB (lanes 16 to 18). GST-12S E1A (lane 18) was added to immunoprecipitates prior to incubation with in vitro-translated, COOH-terminal p300 (amino acids 1240 to 1906). The control immunoprecipitate that lacks GST or GST-12S E1A is shown in lane 16. Lane 14 contains 10% of the p300 in vitro translation reaction product. (C) 13S E1A enhances the interaction between cyclin E-Cdk2 and p300 but has no effect on TFIIB-p300 complexes. Immunoprecipitations were performed with a control antibody (lane 20), an antibody to TFIIB (lanes 21 to 25), or antibodies to Cdk2 (lanes 26 to 30). Increasing amounts of control GST (lanes 22, 23, 27, and 28) or GST-13S E1A (lanes 24, 25, 29, and 30), as indicated, were added to immunoprecipitates prior to incubation with in vitro-translated, carboxy-terminal p300 (amino acids 1240 to 1906). The control immunoprecipitates that lack the addition of GST or GST-13S E1A are shown for TFIIB (lane 21) and Cdk2 (lane 26). Lane 19 contains 10% of the p300 in vitro translation reaction product. Ab, antibody.