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P53 deacetylation assay

GD Giulia Dell’Omo
DC Daniela Crescenti
CV Cristina Vantaggiato
CP Chiara Parravicini
AB Aurora Paola Borroni
NR Nicoletta Rizzi
MG Mariangela Garofalo
AP Andrea Pinto
CR Camilla Recordati
ES Eugenio Scanziani
FB Fabio Domenico Bassi
GP Giancarlo Pruneri
PC Paola Conti
IE Ivano Eberini
AM Adriana Maggi
PC Paolo Ciana
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The SIRT1 deacetylase activity was tested on the K382 residue of native p53 present in the protein extract of MDA-MB-231 cells pre-treated with 20 μM etoposide. Briefly, rhSIRT1 (final dilution 1:60 ab156065 #6), 200 μM NAD and 300 μM inhibitors (NSAIDs or exilusind) were added to 15 μg protein extract of MDA-MB-231, incubated for 30 min at room temperature and stopped with the addition of Laemmli’s sample buffer. The entire reaction was loaded on a SDS-PAGE for immunoblot analysis; immunodetection was carried out using specific antibodies recognizing acetylated p53 (K382 residue) or total p53.

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