JNCI Journal of the National Cancer Institute

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• Supplementary Figure 1 - Western blot analysis of p53 and β-actin protein levels as a function of time of treatment with Bortezomib. Immunoblotting of lysates from wild type (wt, a, SH-SY5Y and b, IMR-32) or mutant (c, LAN-1 and d, SK-NBE2(c)) p53 Neuroblastoma cell lines were performed after 6, 12 and 24 hours after treatment with 20 nM of Bortezomib. Briefly, 40 μ-g samples of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis, with prestained molecular weight markers run in parallel to identify p53 (53 Kd) proteins. The resolved proteins were blotted onto nitrocellulose membranes and the mAb anti-p53 (clone PAb 1801, BD Biosciences, Palo Alto, CA) was used to localize the corresponding polypeptide on the blots. Peroxidase-conjugated goat anti-mouse antibody was used as secondary antibody (BD Biosciences). Immune complexes were visualized with the use of an enhanced chemiluminescence system (Amersham International, Little Chalfont, Buckinghamshire, UK) according to manufacturer’s instructions. To determine whether approximately equal amounts of protein were loaded in the samples, the stripped nitrocellulose membranes were stained with a mAb anti-human β-actin (clone C4; Boehringer Mannheim Inc., Mannheim, Germany). Levels of p53 were quantified by scanning densitometry of the autoradiography films and normalized to β-actin levels.