Isolation of p53 Complexes from GBM Cells

Protocols to isolate p53 complexes from GBM cells (U87MG line) have been previously described.^{13 (link)} Briefly, using the NE-PER kit (Thermo Fisher Scientific), cytoplasmic and nuclear fractions were separated from GBM cell pellets (~100 μL/pellet). The nuclear fraction was incubated with Ni–NTA (nickel–nitrilotriacetic acid) agarose beads (Qiagen) for 1 h at 4 °C. Beads were then washed with five bed volumes of 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-salt buffer (20 mM HEPES, (pH 7.2), 140 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, and 5 mM imidazole). Complexes were eluted with the same HEPES buffer, supplemented with 60 mM imidazole. Fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) denaturing gels through SimplyBlue SafeStain (Invitrogen) or western blot.^{13 (link),14} Western blot analysis used antibodies against p53 (DO-1; Santa Cruz Biotechnology, sc-126) to detect complexes.