Enhanceosome–CBP RNA PolII holoenzyme interactions are required at the early steps of PIC assembly. (A) Immobilized IFN-β enhancer–promoter DNA with (lane 3) or without (lane 2) the enhanceosome was preincubated with HNE followed by washes and Western blot analysis by using antibodies against the depicted proteins. (B) An immobilized IFN-β enhancer–promoter DNA with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) the enhanceosome was preincubated with a complete nuclear extract (lanes 1 and 2) or with the CBP/p300-depleted extract (lanes 3–8), followed by wash and addition of NTPs to initiate transcription. In lanes 5 and 6, the CBP/p300 immunoprecipitate was added before wash whereas in lanes 7 and 8, the immunoprecipitate was added after washing the beads. (C) Same as in A except a CBP-depleted HNE (lanes 2, 5, and 6) was used in parallel with the complete HNE (lanes 1, 3, and 4).