P53k382ac

Cells were dissolved in lysis buffer containing protease inhibitors (10 mM Tris HCl pH 8.0, 150 mM NaCl, 10 mM NaF, 1% NP40, 1 mM PMSF). The proteins were then subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22 μm polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in Tris buffer pH 8.0/0.15% Tween 20 (TBS-T) at room temperature for 1 h and then incubated with primary antibodies diluted in TBS-T, including antibodies against p53 (Santa Cruz), p53K382ac (Abcam), SIRT1 (Abcam), p53K381ac (Abcam), and ERK (Santa Cruz), overnight at 4°C. After three washes in TBS-T, the membranes were incubated with corresponding secondary antibodies, horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare) and horseradish peroxidase-conjugated anti-mouse IgG (GE Healthcare), for 1 h at room temperature. Immunocomplexes were visualized using Amersham EC Western Blotting Detection Reagents (GE Healthcare). The molecular weight of proteins was estimated with pre-stained protein markers (Fermentas). Densitometry analysis was performed using ImageJ software.