FIG. 4.

Affinity chromatography of pooled ∼2-MDa fractions from gel filtration chromatography. Nuclear extracts from uninfected or infected cells were fractionated by Sepharose CL-2B gel filtration chromatography. Fractions surrounding the 2-MDa peak were pooled (fractions indicated in Fig. 2 and 3) and chromatographed on GST and GST-TFIIS columns. Bound proteins were eluted sequentially with buffers containing 0.3 and 0.5 M NaCl. Eluates were concentrated and analyzed by immunoblotting with antibodies that recognize the large subunit of RNAP II, the p56E subunit of TFIIE, the TBP subunit of TFIID, and the cdk7 subunit of TFIIH. Each blot contained a lane of uninfected (A) or infected (B) nuclear extract (20 μg each) to indicate positions of GTFs, as well as a lane containing one-quarter of the flowthrough (Fl-Thru) and a lane containing one-half of the wash.