FIG. 3.

p53-T complex (p53/T) is incapable of stimulating DA complex assembly. (A) p53-T complex fails to induce DA complex formation. Mg-EMSA reactions were carried out as described in Materials and Methods. The amount of each factor added to the Mg-EMSA reactions corresponds to 1 μl of TFIID, 20 ng of recombinant TFIIA (rTFIIA), 50 or 100 ng of p53, and 3 or 6 μl of p53-T complex (containing 100 or 200 ng of p53). The positions of the DA complex, p53-T–DNA, and p53-DNA are indicated on the right. (B) Experiments were performed as for panel A but in the presence of 100 ng of anti-TBP antibody (α-TBP, N-12). (C) p53 purified from mouse cells is capable of stimulating DA complex assembly. Mg-EMSA reactions were carried out as described. The amount of each factor added to the Mg-EMSA reactions (as indicated above each lane) corresponds to 1 μl of TFIID, 20 ng of rTFIIA, 100 ng of p53, and 100 ng of p53-T without T antigen (p53/T −T). The position of the DA complex is indicated on the left. (D) A silver-stained SDS-PAGE gel is shown. Lane 1, 200 ng of HA-tagged human p53; lane 2, 100 ng of HA-tagged ΔN92; lane 3, 1 μl of purified p53-T antigen complex corresponding to 30 ng of p53; lane 4, 1 μl of purified p53-T complex without T antigen corresponding to 30 ng of p53. The sizes (in kilodaltons) of molecular mass standards are indicated on the left.