After treatments, cells were collected and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 250 mM NaCl, and 0.1% Triton, and completed with protease (ThermoScientific, A32953) and phosphatase inhibitors (ThermoScientific, 88667). Total protein extracts were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter, and subjected to immunoblot assay. Following primary antibodies were used: H3K9Ac (Millipore, 07–352); H3K56Ac (Abcam, ab76307); SIRT6 (Novus Biologicals, NBP1-30101); LC3B (Sigma-Aldrich, L7543); total AMP-activated protein kinase (AMPK; Cell Signaling, 2532) and phospho AMPK (Thr172; Cell Signaling, 2535); total (Calbiochem, ST1521) and phospho ULK1 (Ser555 and Ser757; Cell Signaling, 5869 and 6888 respectively); total mTOR (Cell Signaling, 4517) and phospho mTOR (ser2448; Cell Signaling, 2971); Beclin-1 (Cell Signaling, 3738); ATG5 (Cell Signaling, 2630); PARP1 (BD Pharmingen, 551025); β-actin (Sigma, A5441); HSP72/73 (Calbiochem, 386032); and H3 (Abcam, ab1791). Following secondary antibodies were used: Goat anti-mouse or anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugated antibodies (Biorad, 1706516 and 1706515 respectively). Densitometry was performed using ImageJ software.
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