TP53 is occluded from binding the nucleosome core region. (A) Nucleosomes containing 16 different sequences were bound to increasing amounts of TP53 protein and separated by native PAGE. Lanes contain the following: (1) 0.16 pmol nucleosomes; (2–5) 0.16 pmol of nucleosomes with 36, 71, 142, or 286 nM of TP53 (0.25, 0.5, 1, or 2 pmol). Nucleosome and the major supershift bands are indicated (Ss-1, Ss-2, Ss-3). (B–D) Relative supershift for each nucleosome is determined by counting the frequency of each sequence within the supershift and comparing it to nonspecific binding to the 601 sequence. This value is then normalized to the input ratios of nucleosomes (see Equation 1). Error bars, SEM; P-values are shown comparing each nucleosome sequence at a specific TP53 concentration to background levels in the input lane. (B) First supershift band (Ss-1). (C) Second supershift band (Ss-2). (D) Third supershift band (Ss-3). (E) Nucleosome sequences that are lost from the nucleosome band were quantified relative to the input nucleosomes. Strongly bound nucleosomes will generate negative relative shifts.
