TERT Promoter DNA-Protein Interaction

Biotinylated DNA baits consisting of the 2^nd Sp1 binding site in the TERT promoter or a mutated sequence were prepared as previously described [51 (link)]. For primer sequences see Table 4. Sp1 WT or Sp1 mut DNA was immobilized on 250 μg paramagnetic streptavidin beads (Dynabeads MyOne C1, Life Technologies) on a rotation wheel for 30 min at room temperature. Subsequently, baits were incubated with 100 μg of nuclear extract in PBB buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 0.5% Igepal CA-630 (Sigma)) while rotating for 2 h at 4°C. 20 μg sheared salmon sperm DNA (Ambion) was added as a competitor for DNA binding. After three washes with PBB buffer, bound proteins were eluted in 2× Laemmli buffer (Sigma-Aldrich), boiled for 5 min at 95°C and separated on a 4–12% NuPAGE Novex Bis-Tris precast gel (Life Technologies).