Anti acetyl p53 lys382

Whole cell protein extracts were prepared according to Laemmli (1970 (link)). The primary antibodies used were: anti-ATM (1:500), anti-phospho-ATM Ser1981 (1:500), anti-p300 (1:500) (Abcam, Cambridge, UK), anti-GAPDH (1:50000) (Millipore, Darmstadt, Germany); anti-p21WAF1/Cip1 (1:500) (Sigma-Aldrich, St. Louis, USA); anti-p53 (1:500), (Santa Cruz Biotechnology, Santa Cruz, USA); anti-ATR (1:500), anti-phospho-ATR Ser428 (1:500), anti-phospho-p53 Ser15 (1:250), anti-acetyl-p53 Lys382 (1:200), anti-SIRT1 (1:250), anti-phospho-SIRT1 Ser47 (1:250), anti-p38 MAPK (1:500), anti-phospho-p38 MAPK Thr180/Tyr182 (1:500), anti-phospho-MAPKAPK-2 Thr334 (1:500), anti-AMPKα (1:500), anti-phospho-AMPKα Thr172 (1:1000), anti-ACC (1:500), anti-phospho-ACC Ser79 (1:1000), anti-mTOR (1:500), anti-phospho-mTOR Ser2448 (1:500), anti-phospho-S6 Ser235/236 (1:1000) (Cell Signaling Technology, Denvers, USA), anti-Rb (1:250) (NeoMarkers, Fremont, USA). The respective proteins were detected after incubation with one of the horseradish peroxidase-conjugated secondary antibodies (1:2000) (Dako, Glostrup, Denmark), using an ECL system (Thermo Scientific, Rockford, USA), according to the manufacturer’s instructions.

Proteins were extracted from each sample, at 5 days and 2 weeks after UVB exposure. Total protein (15 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted with anti-H2A histone family member X (H2AX), anti-γ-H2AX, anti-p53, anti-phospho-p53 (Ser15), anti-acetyl-p53 (Lys382), and anti-p21CIP1 antibodies (Cell Signaling Technology, Danvers, MA, USA), and with anti-TYR, anti-TYRP1, anti-TYRP2, and anti-GAPDH antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), respectively.

Equal amounts of protein were run on 4%–20% SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp. Billerica, Massachusetts, USA). The blots were probed with the following primary antibodies: anti-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetyl-p53 (Lys382; Cell Signaling, Beverly, MA, USA), anti-FUT8 (Novus Biologicals, Littleton, CO, USA), and anti-ß-actin (Santa Cruz Biotechnology). Subsequently, the membranes were incubated with goat anti-mouse- or goat anti-rabbit horseradish peroxidase and visualized by enhanced chemiluminescence (GE Healthcare, Amersham, UK). The blots were quantified using LAS-4000UV mini and Multi Gauge software (Fujifilm, Tokyo, Japan).