35s methionine labeling mix

The relative amount of protein synthesis was determined by measuring the amount of incorporation of [³⁵S]-methionine into the protein. Briefly, cells were precultured in methionine-free medium (supplemented with 10% dialyzed FCS) for 3 h, followed by incubation with 3.5 μCi of [³⁵S]-methionine-labeling mix (PerkinElmer) per 8 × 10⁵ cells for 6 h as indicated. After the treatment, cells were washed twice with ice-cold PBS and lysed in RIPA buffer. Then, 50 μl of each lysate was added to 1 ml of Liquid Scintillation Cocktail solution (Beckman Coulter, Brea, CA, USA) and the amount of incorporated radioactivity was determined by liquid scintillation counting.

For immunoprecipitation, cells were treated for 4 h with Etoposide and/or Roc-A in the absence or presence of 100 nM Bortezomib (Enzo Life Sciences). In the case of metabolic pulse-labeling experiments, treatment was followed by adding 100 μCi/ml of [³⁵S]-methionine-labeling mix (PerkinElmer, Waltham, MA, USA) to the medium for 0–15 min. Subsequently, cells were washed in ice-cold PBS, lysed in IP buffer (20 mM Tris-HCl, 5 M NaCl, 2 mM EDTA, 1% Triton X-100, protease inhibitors) and centrifuged (10 000 × g, 20 min) to clear lysates. Aliquots were taken for input control and lysates were incubated overnight with sepharose-coupled protein A beads, anti-p53 antibody (FL-393; Santa Cruz) or isotype control antibody (Sigma-Aldrich). Two wash steps with IP buffer preceded boiling of beads in denaturing sample buffer at 95°C for 5 min. Incorporation of [³⁵S]-methionine into p53 protein was detected by the phosphoimaging system FLA-7000 IR (Fujifilm Europe GmbH, Düsseldorf, Germany).