Figure 3. p53 binds to the BCL6 p53RE in vitro and in vivo. (A) p53 in vitro binding to the BCL6 p53RE was assayed by EMSA. Wild-type (lanes 2-6, 8) and mutant (lane 7) p53 proteins were incubated with the specified oligos. Oligo A, wild-type BCL6 p53RE (lanes 1-3, 5-7); Oligo B, like oligo A without the extra guanosine in the middle of the first decamer of the BCL6 p53RE (lane 4); Oligo C, mutated BCL6 p53RE (lane 8); supershift with anti-p53 antibody (lane 3); competition with a 50-fold or 500-fold excess of unlabeled oligo A (lanes 5 and 6, respectively). Ab indicates antibody. ×'sA indicates an excess of unlabeled Oligo A. (B) p53 in vivo binding to the BCL6 p53RE was assayed by ChIP in lymphoblastoid B cells. Immunoprecipitation of p53 protein-DNA complexes was done with anti-p53 antibody. Negative controls were chromatin immunoprecipitated with an irrelevant antibody (anti-GFP) and normal mouse IgG, as well as samples to which no antibody was added (no Ab). Positive control was chromatin immunoprecipitated with anti-RNA polymerase II antibody. Primers specific for the CDKN1A promoter and GAPDH exon 4 served as positive and negative controls, respectively. Input indicates 0.1% of the sonicated chromatin before immunoprecipitation.
Figure 3.

p53 binds to the BCL6 p53RE in vitro and in vivo. (A) p53 in vitro binding to the BCL6 p53RE was assayed by EMSA. Wild-type (lanes 2-6, 8) and mutant (lane 7) p53 proteins were incubated with the specified oligos. Oligo A, wild-type BCL6 p53RE (lanes 1-3, 5-7); Oligo B, like oligo A without the extra guanosine in the middle of the first decamer of the BCL6 p53RE (lane 4); Oligo C, mutated BCL6 p53RE (lane 8); supershift with anti-p53 antibody (lane 3); competition with a 50-fold or 500-fold excess of unlabeled oligo A (lanes 5 and 6, respectively). Ab indicates antibody. ×'sA indicates an excess of unlabeled Oligo A. (B) p53 in vivo binding to the BCL6 p53RE was assayed by ChIP in lymphoblastoid B cells. Immunoprecipitation of p53 protein-DNA complexes was done with anti-p53 antibody. Negative controls were chromatin immunoprecipitated with an irrelevant antibody (anti-GFP) and normal mouse IgG, as well as samples to which no antibody was added (no Ab). Positive control was chromatin immunoprecipitated with anti-RNA polymerase II antibody. Primers specific for the CDKN1A promoter and GAPDH exon 4 served as positive and negative controls, respectively. Input indicates 0.1% of the sonicated chromatin before immunoprecipitation.

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