FIG. 3.

Effect of ICP4 on the binding of purified HA-TFIID to immobilized gC promoters. (A) Silver-stained SDS-PAGE gel of HA-tagged TFIID purified from Hela cells expressing HA-tagged TBP. TAF subunits, according to their estimated molecular weights, and TBP are shown on the left side of the gel. (B) Effect of ICP4 concentration on the binding of HA-TFIID to immobilized gC promoter templates. HA-TFIID (4 μl; (approximately 10 fmol) was incubated with 100 fmol of immobilized gC promoter in the presence of 75 to 1,200 fmol of purified ICP4 protein or in the absence of ICP4 in a total volume of 300 μl. After incubation at 30°C, nucleoprotein complexes bound to immobilized promoter templates were isolated and analyzed as described in Materials and Methods. (C) Effect of the HA-TFIID concentration on its recruitment to immobilized gC promoter templates in the presence of ICP4. HA-TFIID (from 2 to 8 μl) was incubated with 100 fmol of immobilized gC templates in the presence of approximately 300 fmol of ICP4 or in the absence of ICP4 in a total volume of 300 μl. gC-bound ICP4, TAF250, TAF150, and HA-TFIID were isolated and analyzed as described in Materials and Methods.