FIG. 1.

PC4 activates sequence-specific DNA binding of p53. (A and B) Recombinant proteins were used in different experiments. (A) Lane 1, 300 ng of recombinant PC4 was analyzed by SDS-15% PAGE. Lane 2, authenticity of the PC4 was analyzed by Western blotting using anti-PC4 polyclonal antibody N17. (B) Lane 1, 100 ng of recombinant FLAG-tagged p53 was analyzed by SDS-10% PAGE. Lane 2, authenticity of the p53 was analyzed by Western blotting using anti-p53 monoclonal antibody PAb421. (C and D) Effect of PC4 in p53-mediated DNA binding.. (C) Three nanograms of a ³²P-labeled oligonucleotide containing a p53 binding site was incubated with 50 ng of p53 either in the absence of PC4 (lane 2) or in the presence of 50 ng (lane 3), 100 ng (lane 4), 200 ng (lane 5), or 300 ng (lane 6) of PC4. The ³²P-labeled oligonucleotide was also incubated with 50 ng (lane 7), 100 ng (lane 8), 200 ng (lane 9), and 300 ng (lane 10) of PC4 alone. Lane 1 contains the ³²P-labeled oligonucleotide alone. (D) EMSA was done as described in the text in the presence of p53 (50 ng) either alone (lane 2) or in combination with PC4 (300 ng) (lane 3) and competitor DNA (100× [lanes 4 and 6] and 200× [lanes 5 and 7]) containing wild-type (WT) (lanes 4 and 5) and mutant (mt) (lanes 6 and 7) p53 binding sites. Lane 1 contains ³²P-labeled oligonucleotide alone.