Research Paper Volume 1, Issue 1 pp 58—67

Figure 3. ΔNp63α modulates binding of Sp1 to Sp1 DNA-binding region by decreasing the SIRT1 protein levels and deacetylation of p53. Chromatin immunoprecipitation assay (X-ChIP). Mouse epidermal keratinocytes (5x10⁷ cells) expressing heterozygous p63-/+ (samples 1-5) and overexpressing ΔNp63α(samples 6-10) were treated with control media (samples 1 and 6), SIRT1 inhibitor (Sirtinol, 100 μg/ml for 24 h; samples 2 and 7), SIRT1 shRNA (samples 3 and 8), p53 shRNA (samples 4 and 9), and shSp1 RNA (samples 5 and 10). The protein-DNA complexes were precipitated with a primary antibody for Sp1. As negative controls, we used immunoglobulins (IgG) from rabbit (IgG-R) or mouse (IgG-M) sera. The mTERT-derived Sp1 promoter region using the following primers: sense (SP1), 5'-CTCACTGTCTGTGCAACCACAGCAGCTG-3' (position-363), and antisense (AP2), 5'-AGAGCACCGCGGGGCAACGAGGAGCGCG-3' (position +143) giving raise to a 506 bp PCR product. The PCR products were run on 2% agarose gels and visualized by ethidium bromide staining.