Immunoblot analysis of autophagy and epigenetic markers

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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Iachettini S., Trisciuoglio D., Rotili D., Lucidi A., Salvati E., Zizza P., Di Leo L., Del Bufalo D., Ciriolo M.R., Leonetti C., Steegborn C., Mai A., Rizzo A, & Biroccio A. (2018). Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells. Cell Death & Disease, 9(10), 996.

Publication 2018

phosphatase inhibitors H3K9Ac H3K56Ac total mTOR Beclin-1 PARP1 β-actin

Actin After treatments Amp activated protein kinase Anti antibodies Antibodies Beclin 1 Biologicals Buffer Cells Densitometry Edta Goat Horseradish peroxidase Immunoblot assay Immunoglobulin g Inhibitors Mouse Mtor Nacl Nitrocellulose Novus Parp1 Phosphatase Protease Protein Rabbit Sds polyacrylamide gel electrophoresis Sirt6 Tris Ulk1

Corresponding Organization :

Other organizations : Istituti di Ricovero e Cura a Carattere Scientifico, Sapienza University of Rome, University of Rome Tor Vergata, University of Bayreuth

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Protocol cited in 3 other protocols

Variable analysis

independent variables

Treatments applied to the cells

dependent variables

Protein expression and post-translational modifications (e.g., H3K9Ac, H3K56Ac, SIRT6, LC3B, AMPK, ULK1, mTOR, Beclin-1, ATG5, PARP1)
Densitometry measurements of protein levels

control variables

50 mM Tris-HCl (pH 7.5) buffer
5 mM EDTA
250 mM NaCl
0.1% Triton
Protease and phosphatase inhibitors
SDS-polyacrylamide gel electrophoresis
Nitrocellulose filter transfer
Primary and secondary antibodies used for immunoblot assay
β-actin and H3 as loading controls

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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