Acetyl lysine

Blood inside organs were removed by rinsing tissues with PBS. Tissues were snap-frozen under liquid nitrogen. Tissues were homogenized in RIPA buffer (Sigma) with protease (Roche), deacetylase (nicotinamide, Tricostatin A) and phosphatase inhibitors (Roche)^{7 (link)}. Protein concentrations of samples were determined by Lowry assay and each amount of protein (30–50 ug per sample) were loaded for SDS-PAGE. Antibodies from the following companies were used for Western blot analysis: Phospho-H2Ax (1:500, NBP1-19255-Novus Biological), PAR (1:500, AM80100UG-Millipore), actin (1:5000, sc-8432-Santa Cruz), nitrotyrosine (1:500, 06-284-Millipore) acetyl-lysine (1:1000, 9441-Cell signaling), HIF1a (1:400, 14179-Cell Signaling), GLUT1 (1:500, ab652-Abcam), PDK4 (1:1000, 3820-Cell Signaling), LDHA (1:1000, 3582-Cell Signaling), SDHA (1:10000, ab14715-Abcam), Ndufs4 (1:1000, ab87399-Abcam,), SOD2 (1:1000, ab16956-Abcam) SOD2-Ac (1:1000, ab137037-Abcam), GDH (1:1000, 12793-Cell Signaling). Blots were blocked in 5% BSA-TBST. Antibodies were diluted in 5% BSA-TBST. Protein bands were visualized with chemiluminescence assay (Pierce) with secondary antibodies coupled with HRP. The protein abundance was analyzed by densitometry with ImageJ. SDHA or actin were used as a loading control for quantification.

CAPE was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Retinol was purchased from Cayman (Ann Arbor, MI, USA). Antibodies for phospho-p38, phospho-JNK, phospho-c-Jun, acetyl lysine, H3K9ac, and Histone H3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for p38, JNK, c-Jun, and vinculin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody for MMP-1 was obtained from R&D systems (Minneapolis, MN, USA).

293 cells were transiently co-transfected with FLAG-BCL9, together with histone acetyltransferases (P300/CBP/pCAF), along with FLAG-SIRT2-WT or FLAG-SIRT2-H187Y, and cultured with 0.5 μM TSA for 12 h. Cells were harvested in PBS and lysed for 20 min on ice in IP lysis buffer (0.75% CHAPS, 10% glycerol, 150 mM NaCl, 50 mM Tris pH 7.5) freshly supplemented with protease inhibitors and 1uM TSA. Lysates were clarified by centrifugation (13,000 rpm, 15 min at 4 °C), the supernatants were then collected and diluted by same volume of dilution buffer (10% glycerol, 150 mM NaCl, 50 mM Tris pH 7.5) to adjust the CHAPS concentration to 0.375%. Protein concentration was then determined and lysates of 2 mg protein were used for immunoprecipitation reaction, protein lysates were immunoprecipitated using anti-FLAG M2 agarose (Sigma). The immunocaptured proteins were analyzed for deacetylation by immunoblotting with anti-FLAG and anti-acetyl antibody. The antibodies used were as follows: tubulin (Sigma; T6074), FLAG (Santa Cruz, sc-51590), acetyl lysine (Cell Signaling, 9441).

Whole-cell lysate prepared as described [31 (link)] was used to determine the levels of proteins. Antibodies used in this study include those against XPA (Kamiya), XPB-XPD (Santa Cruz Biotechnology, Santa Cruz, CA, USA), XPE, p-p53, p-CHK1, GAPDH, SIRT1, acetyl-lysine (Cell Signaling Technology), XPF, and XPG (both Abcam, Cambridge, UK). For immunoprecipitation of XPA, 1 mg of whole-cell lysate was incubated with 1 μg of anti-XPA conjugated to Protein A/G-agarose beads (Sigma, St. Louis, MO, USA) for 12 h at 4°C with rotation. After washing with lysis buffer, proteins were eluted from the beads by boiling in SDS sample buffer and resolved on 10% SDS-polyacrylamide gels. For detection of XPA acetylation, anti-acetyl-lysine was employed.