FIG. 6.

Fusion proteins GST-2T-TBP.184-220, and GST-2T-T.181-205 stimulate SV40 DNA replication in vitro and inhibit the interaction between T antigen and TFIID. (A) One microgram of purified full-length T antigen was incubated with 10 μl of 12CA5-Sepharose beads preincubated with HeLa control extract (lane 2) or eTFIID immobilized on 12CA5-Sepharose beads in the absence (lane 3) or presence of the indicated amounts of GST fusion proteins (lanes 4 to 9). After the beads were washed, the bound proteins were eluted by boiling in sample buffer and separated by SDS-PAGE (12.5% polyacrylamide gel). T antigen was detected by immunoblotting with the monoclonal anti-T antigen antibody Pab419. Lane 1 shows 0.1 μg of the input T antigen as a marker. Positions of T antigen (T Ag) and immunoglobulin heavy and light chains (IgH and IgL) are shown on the right. (B) Increasing amounts of GST, GST-2T-TBP.184-220, or GST-2T-T.181-205 were added to an SV40 DNA replication reaction containing 190 μg of undepleted S100 extract from 293S cells. Replication activity was quantitated by measuring the incorporation of radiolabeled nucleotides into DNA.