The project described here was supported by a grant from NIH/NCI (NCI-R01CA182506-01A1) as well as by the Robert H. Lurie Comprehensive Cancer Center - The Lefkofsky Family Foundation/Liz and Eric Lefkofsky Innovation Research Award to A.V. We would like to thank members of the laboratory (Carol O&#39;Callaghan and Elizabeth Anne Wayne) for critical reading and editing this manuscript.

1. In Vitro Deacetylation Assay
<ol>
	<li>Purification of SIRT2
	<ol>
		<li>Prepare a 10 cm culture dish of HEK 293T cells cultured in 8 ml DMEM with 10% FBS and antibiotics and grown in a 37 &#176;C, 5% CO2 tissue culture incubator to be about 70% confluent the next day.</li>
		<li>The next day, co-transfect 4 &#181;g pCDH-puro-GFP-SIRT2-Flag (lentivector made in our lab), 4 &#181;g pCMV- dR8.2 dvpr (packaging vector) and 0.5 &#181;g pCMV-VSV-G (envelope vector) 18 using polyethylenimine...

<ol>
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Cell Physiology. 307, C763-C773 (2014).</li><li>Allfrey, V. G., Faulkner, R., Mirsky, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+and+Methylation+of+Histones+and+Their+Possible+Role+in+the+Regulation+of+Rna+Synthesis.">Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 51, 786-794 (1964).</li><li>Shahbazian, M. D., Grunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+site-specific+histone+acetylation+and+deacetylation.">Functions of site-specific histone acetylation and deacetylation.</a> Annual Review of Biochemistry. 76, 75-100 (2007).</li><li>L'Hernault, S. W., Rosenbaum, J. 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Recent high throughput proteomic studies have established acetylation as a widespread PTM found not only in nucleus but also in cytoplasm and mitochondria 7,8,21-23. Taking into account the likelihood that many more acetylated proteins and sites might have been not detected due to several reasons, such as specificity of the anti-Ac-K antibodies used, the low abundance of the acetylated proteins, and the transient nature of the PTM, it is safe to predict that more acetylated proteins remain to be discovered in ...

Post-translational modifications (PTMs) regulate cell signaling networks allowing cells to rapidly respond to internal and external signals. Over the last few decades, many different PTMs playing a pivotal role in diverse processes have been identified but only a few have been studied extensively, such as phosphorylation, acetylation and ubiquitination 1-3. Focusing on acetylation, Allfrey et al. were the first to propose a role for histone acetylation in regulating gene transcription about 50 years ago 4. Research in this field has revealed that histone lysine acetylation modulates chromatin condensation and it is considered to be an epigenetic mark as part of the histone code 5. Although it took a long time until the discovery of tubulin as the first non-histone acetylation target 6, it is well established now that hundreds of eukaryotic proteins beyond histones can be acetylated and lysine acetylation has been recognized as a wide-spread PTM that may rival phosphorylation and ubiquitination in its prevalence 7-9. Interestingly, non-histone acetylated proteins can be signaling molecules in the cytoplasm, transcription factors in the nucleus, and metabolic enzymes in mitochondria, highlighting the significance of acetylation in regulating a plethora of cellular processes. 
The acetylation status of a protein depends on the coordinated and opposing function of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) which add and remove acetyl groups from proteins. The reversible acetylation of lysine, which involves neutralization of a positive charge 10, alters protein structure and it seems very likely to also alter enzymatic function in several cases 11-13. Focusing on KDACs, 18 proteins have been identified in the human and mouse genomes 14-16. Among them, mammalian sirtuins (also called class III histone lysine deacetylases) which are distinct from other members as they require NAD+ for their enzymatic function, have attracted extensive interest in this research field 16. In mammals, seven sirtuins (SIRT1-7) have been identified, each of them sharing a conserved 275-amino-acid catalytic core domain, which are mainly categorized according to their subcellular localization to the nucleus (SIRT1, 6, and 7), mitochondria (SIRT3, 4, and 5), or cytoplasm (SIRT2). SIRT1-3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity, SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity but are involved in other types of acylations 17. In accordance with the significance of acetylation as a regulatory PTM modification involved in several cellular functions, sirtuins have also been implicated in a wide range of processes. After the first breakthrough studies establishing the role of sirtuins in life span extension, it has been shown that they are involved in diverse cellular functions including DNA repair, maintenance of genomic instability, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks and metabolism, as well as contributing to the initiation and/or progression of age-related diseases such as cancer, neurodegeneration and type 2 diabetes 15,16.
Despite the significant progress in the field of sirtuin biology, more work remains to unravel undiscovered roles and functions through the identification of novel substrates. This is evidenced more emphatically by recent advances in high-resolution mass spectrometry (MS) based proteomics which have significantly increased the number of proteins found to be acetylated but most importantly have identified several different acetylated lysines in each protein, arguing that acetylation may be as wide-spread as other PTMs such as phosphorylation 7,8,17. Taking into consideration that specific deacetylases have not yet been identified for most of these acetylated proteins-substrates, it is reasonable to suggest that both in vitro and in vivo deacetylation assays are needed to confirm and establish an acetylated protein as a legitimate substrate of a specific deacetylase. In the experimental protocols described below, details will be given on how to perform both in vitro and in vivo deacetylation assays using SIRT2 as the specific deacetylase.

<table border="0" cellpadding="0" cellspacing="0" width="1543">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">cell culture dishes</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">T1110 and T1115</td>
			<td style="width:728px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">pCDH-puro-GFP lentiviral vector</td>
			<td style="width:239px;">System Biosciences</td>
			<td style="width:119px;">CD513B-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV- dR8.2 dvpr (packaging vector)</td>
			<td style="width:239px;">Addgene</td>
			<td>8455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV-VSV-G (envelope vector)</td>
			<td style="width:239px;">Addgene</td>
			<td align="right">8454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylenimine (PEI)&#160;</td>
			<td>Polysciences Inc.</td>
			<td align="right" style="width:119px;">24885</td>
			<td>other transfection reagents can be used as well. PEI is cost effective and very efficient in transfecting 293T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22&#956;m filters</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">F5512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polybrene</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H9268</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">fluorescent microscope</td>
			<td style="width:239px;">Carl Zeiss MicroImaging Inc.</td>
			<td style="width:119px;">Axiovert 200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:239px;">Invivogen</td>
			<td style="width:119px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:239px;">Corning</td>
			<td style="width:119px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag antibody</td>
			<td style="width:239px;">Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">M9272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">EGTA</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">34596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protease inhibitors coctail 100x</td>
			<td style="width:239px;">Biotool</td>
			<td style="width:119px;">B14001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichostatin A (TSA)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T8552</td>
			<td>selective inhibitor of class I and II histone deacetylases (HDACs) but not class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag agarose beads</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:119px;">5417R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">rotator</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td style="width:119px;">415220Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">filter tubes</td>
			<td style="width:239px;">Millipore</td>
			<td>UFC30HV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flag peptide&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td>F3290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivaspin Centrifugal Concentrator</td>
			<td>Sartorius Stedim Biotech S.A.</td>
			<td>VS0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain solution&#160;</td>
			<td style="width:239px;">Invitrogen</td>
			<td style="width:119px;">LC6060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE LDS sample buffer (4x)</td>
			<td style="width:239px;">Life Techologies</td>
			<td style="width:119px;">NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Ac-K antibody</td>
			<td style="width:239px;">Cell Signaling</td>
			<td style="width:119px;">9441</td>
			<td>several anti-Ac-K antibodies are available. In our hands, the Cell Signaling antibody exhibits the highest sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl pH 7.5</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T5941</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NAD+</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">N0632</td>
			<td>required cofactor for sirtuins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nicotinamide (NAM)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">72340</td>
			<td>selective inhibitor class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pLKO.1 lentiviral vector&#160;</td>
			<td style="width:239px;">Addgene</td>
			<td style="width:119px;">8453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SIRT2 si RNA&#160;</td>
			<td style="width:239px;">Qiagen</td>
			<td style="width:119px;">GS22933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-SIRT2 antibody</td>
			<td style="width:239px;">Proteintech</td>
			<td style="width:119px;">15345-1-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bradford protein assay</td>
			<td style="width:239px;">BIO-RAD</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti Ac-K agarose beads</td>
			<td style="width:239px;">Immunechem</td>
			<td>ICP0388</td>
			<td></td>
		</tr>
	</tbody>
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deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

In order for a protein to be considered as a legitimate deacetylation target for any enzyme with deacetylation activity, both in vitro and in vivo deacetylation assays need to be performed to establish the interplay between the deacetylase and its substrate. For the in vitro deacetylase assay, the purification of both the deacetylase and the acetylated protein substrate is required before the assay can be done. Here we use the cytoplasmic sirtuin SIRT2 as the sp...

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Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

The overall goal of this protocol is to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins. Despite the increasing number of proteins found to be acetylated, arguing for a widespread presence of acetylation, specific enzymes regulating these modification have not yet been identified for most of these proteins. The main advantage of these assays is that by establishing a protein as a legitimate substrate of a specific deacetylase, novel roles and functions of the deacetylase can be unraveled.Though this method can provide insight into the identification of sirtuin 2-specific substrate, it can also be applied, with minor modifications, to on other members of the sirtuin family. To begin this procedure, prepare a 10 centimeter culture dish of HEK 293 T cells in eight milliliters of DMEM, with 10%FBS in antibiotics so that the cells will be about 70%confluent after growing overnight. On the following day, cotransfect the cells with the following:four micrograms of lentivector, expressing GFP and FLAG-tagged SIRT2, four micrograms of packaging vector and 0.5 micrograms of envelope vector.Use polyethylenimine, or PEI, at a ratio of three microliters of PEI per microgram of DNA. 12 hour after transfection, change the medium. 36 to 48 hours later, collect the supernatant.Remove debris by centrifugation at 1, 000 g for five minutes at four degrees Celsius. After filtering through a 0.22 micrometer filter, make one milliliter aliquots of SIRT2 lentivirus, and store at negative 80 degrees Celsius. When ready to infect the cells, thaw the SIRT2 lentivirus on ice.Infect a six-well plate of 80 to 90%confluent HEK 293 T cells with one milliliter of lentivirus in the presence of eight micrograms per milliliter of Polybrene. Place the virus-infected cells in a 37-degree Celsius incubator for 24 hours. After 24 hours, replace the medium.48 hours after infection, check the infection efficiency by detecting GFP-positive cells under a fluorescent microscope. If infection efficiency is at least 40 to 50%replace the medium with culture medium containing two micrograms per milliliter of Puromycin to select for stable SIRT2 overexpressing cells. Return the cells to the incubator.The selection for stable SIRT2 overexpressing cells will take about two weeks, and the medium must be changed every three to four days. For SIRT2 purification, prepare at least 10 10-centimeter dishes of FLAG SIRT2-expressing HEK 293 T cells. After 24 hours, remove the culture medium, wash the cells twice in PBS and collect cells by trypsinization, followed by centrifugation at 1, 000 g for 10 minutes at four degrees Celsius.Lyse the cell pellet in 500 microliters of lysis buffer A per dish that includes a protease inhibitor cocktail and one micromolar of TSA, a non-class three histone deacetylase inhibitor. Incubate for 30 minutes at four degrees Celsius under constant rotation. After 30 minutes, centrifuge at 14, 000 g for 15 minutes at four degrees Celsius.Transfer the supernatant to a new tube. Perform immunoprecipitation using an anti-FLAG antibody conjugated to agarose beads. Add 200 to 300 microliters of the anti-FLAG antibody to the protein lysate and incubate at four degrees Celsius with constant agitation overnight.On the following day, collect the immunoprecipitates by centrifugation at 1, 000 g and four degrees Celsius for five minutes. Wash the pellet five times with 10 milliliters of lysis buffer A.After the fifth wash, remove the supernatant and add 500 microliters of a one X FLAG peptide solution that includes a protease inhibitor cocktail and TSA to the agarose beads. Incubate at four degrees Celsius, under constant rotation, for 30 minutes.Collect the supernatant by centrifugation at 6, 000 g for two minutes at four degrees Celsius. Remove the agarose beads from the supernatant by using filter tubes and centrifugation at 15, 000 g at four degrees Celsius for one minute. Lastly, use an ultrafiltration membrane to concentrate the eluted sample until the final protein concentration is one microgram per microliter.Keep purified SIRT2 at negative 80 degrees Celsius. Begin this procedure by preparing 10 10-centimeter culture dishes of HEK 293 T cells, so they will be about 70%confluent the following day. On the following day, cotransfect the cells with five micrograms of a FLAG-tagged plasma infector expressing the protein-substrate and five micrograms of the specific acetyltransferase that can acetylate the protein.If the specific acetyltransferase is not known, use a mixture of known histone acetyltransferases. Use PEI at a ratio of three microliters of PEI per microgram of DNA. Incubate the transfected cells for 12 hours.Then, change the medium and incubate the cells for 24 hours. Treat the cells with one micromolar TSA and two millimolar nicotinamide for 12 hours by replacing the culture medium with medium containing TSA and nicotinamide. This will maximize acetylation levels of the protein-substrate by inhibiting deacetylases.48 hours after transfection, remove the culture medium and wash the cells twice with PBS. Collect the cells by trypsinization, followed by centrifugation at 1, 000 g for 10 minutes at four degrees Celsius. To lyse the cell pellet, add 500 microliters of lysis buffer A per dish, that includes a protease inhibitor cocktail, one micromolar TSA and two millimolar nicotinamide, and incubate at four degrees Celsius, under constant rotation, for 30 minutes.Centrifuge at 14, 000 g for 15 minutes at four degrees Celsius and transfer the supernatant to a new tube. Perform immunoprecipitation using an anti-FLAG antibody conjugated to agarose beads, as described in the text protocol. After concentrating the eluted sample to one microgram per milliliter, keep the purified acetylated protein-substrate at negative 80 degrees Celsius.A critical step in this protocol is to confirm purification of the acetylated protein-substrate by protein, using a protein-specific antibody and anti-acetylation antibody prior to the in vitro deacetylation reaction. Prior to starting the in vitro deacetylation reaction, prepare deacetylation buffer B.Next, prepare three reactions in three different tubes, each in a total volume of 20 microliters, as shown in this table. All three tubes contain purified acetylated protein-substrate and deacetylation buffer B, but tube two also contains purified SIRT2, and tube three also contains purified SIRT2 and NAD plus.To confirm deacetylation of the protein-substrate by SIRT2, an optional fourth reaction can be included, in which a purified deacetylase null mutant of SIRT2 is added instead of the deacetylase active SIRT2. Incubate the reactions at 30 degrees Celsius under constant agitation for three hours. Stop the reactions by adding 20 microliters of two X sample buffer to each tube, and boiling the samples at 95 degrees Celsius for 10 minutes.Subsequently, the reaction mixtures are run on a four to 12%SDS page gel, and the acetylation status of the protein-substrate is detected by Western blotting, using an antibody against acetylated lysine. Using this protocol, wild-type SIRT2 and a deacetylation defective mutant, SIRT2 Hi87Y were purified at a final concentration of one microgram per microliter. Since both proteins are tagged with a FLAG peptide, the purification can be confirmed by immunoblotting, using an anti-FLAG antibody.The protein-substrate and acetylated protein-substrate were also purified. Immunoblotting using an anti-acetyl lysine antibody confirmed increased acetylated levels of the purified protein in cells overexpressing the acetyltransferases. Total proteins were detected using an anti-FLAG antibody.In an in vitro deacetylation assay, when the purified acetylated protein-substrate was incubated with wild-type SIRT2 or the deacetylation defective mutant in the presence of NAD plus, decreased acetylated levels were detected by immunoblotting, using an anti-acetyl lysine antibody in the presence of SIRT2, but not in the presence of the mutant. No deacetylation activity was observed when NAD plus was not included in the reaction mixture, confirming that SIRT2 requires NAD plus for its enzymatic function. Deacetylation was also inhibited when a sirtuin inhibitor, NAM, was added to the reaction mixture, which implies that the decrease in the acetylated levels of the protein is mediated by the deacetylase activity of SIRT2.In order for a protein to be considered as a the deacetylation target for any enzyme, both in vitro and in vivo deacetylation assay need to be performed to establish the interplay between the deacetylase and each substrate. In vivo deacetylation assays in cells under specific experimental conditions can provide further insight regarding the interplay between the deacetylase and the potential substrate in physiological and pathophysiological scenarios, and establish the functional role of the deacetylation event, in a physiological context. Given the increasing interest regarding the role of sirtuins in regulating diverse cellular processes, the described deacetylation assays can be used with slight modifications to identify new substrates for other members of the sirtuin family, regardless of their subcellular localization.Both in vitro and in vivo deacetylation assays carried out by specific deacetylases may be further combined with small-scale mass spectrometry analysis to reveal target lysines on the substrate to distinguish functional acetylation sites from non-regulatory acetylated lysines.

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8.1K ビュー.  Feinberg School of Medicine, Northwestern University. The overall goal of this protocol is to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins. Despite the increasing number of proteins found to be acetylated, arguing for a widespread presence of acetylation, specific enzymes regulating these modification have not yet been identified for most of these proteins. The main advantage of these assays is that by establishing a protein as a legitimate substrat...