Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

LC-MS/MS identification of SIRT2 interaction proteins

ABSTRACT: HEK293T cells transfected with plasmids expressing Flag-Luc or Flag-SIRT2 were lysed in CHAPS lysis buffer (120 mM NaCl, 0.3% CHAPS (Sigma, C900480), 1 mM EDTA, 40 mM HEPES (pH 7.5), and complete protease inhibitor cocktail (Roche)) at 4 °C for 2 h with rotation, and then centrifuged at 12 000 g at 4°C for 30 min. For Co-IP with exogenous proteins, the resulting supernatants were mixed with anti-FLAG Affinity Gel (Sigma, A2220) and rotated overnight at 4 °C. The SIRT2 interacting protein complexes by anti-Flag Affinity Gel were competitively eluted using FLAG peptides and processed for Western blotting analysis.

ORGANISM(S): Macaca Fascicularis

SUBMITTER: Weiqi Zhang

PROVIDER: PXD036693 | iProX | Tue Sep 13 00:00:00 BST 2022

REPOSITORIES: iProX

ACCESS DATA

Json Xml

Similar Datasets

The inner nuclear membrane protein LEMD3-interacting proteins

Project description:HEK293T cells were transfected with the pcDNA3.1 or LEMD3-FLAG plasmid.Forty-eight hours after transfection, the cells were lysed in lysis buffer (50 mM Tris-HCl [pH=7.4], 150 mM NaCl, 2 mM EDTA, and 0.3% NP-40). The cell lysate samples were incubated with anti-FLAG M2 affinity gel (A2220, Sigma‒Aldrich) according to the manufacturer’s instructions. The anti-FLAG M2 affinity gel-bound proteins were eluted with FLAG peptide (F3290, Sigma). The final eluent was collected and resolved on an SDS-PAGE gel. The gel was fixed with a 30% ethanol:10% acetic acid solution, washed with 10% ethanol, and then subjected to LC‒MS/MS. Samples from HEK293T cells transfected with the pcDNA3.1 plasmid were used as the negative control.

2025-08-19 | PXD067510 |

Human Cell 293T BCAT1 IP GC-LC-MS

Project description:To identify the potential interaction protein that interacts with BCAT1 by IP-MS, we established the stable cell line with vector and BCAT1 in AGS. Then ten million cells were collected lysed in ice-cold 0.1% NP-40 buffer (50 mM Na2HPO4, pH 7.5, 150 mM NaCl) with protease inhibitors cocktail (Sigma, p8340). The supernatants were collected by centrifugation and incubated with 10 µL Anti Flag‐beads (Sigma, A2220at 4°C for overnight. After incubation, beads were washed with lysis buffer for 3 times. Precipitates were resuspended in 1 × SDS loading buffer and separated by SDS-PAGE (polyacrylamide gel electrophoresis). Then cut off (about 1cm2) gel for trypsin hydrolysis and collected and dried the peptide for MS analysis (Thermo Fisher Q Exactive).

2024-11-15 | PXD041556 | Pride

Identification of GFI1 target genes in human lung adenocarcinoma cell line A549 [ChIP-seq]

Project description:To investigate the genes regulated by the transcription factor GFI1 in lung cancer cells, we overexpressed GFI1-Flag in A549 cells and performed ChIP-Seq with anti-Flag affinity gel.

2022-09-14 | GSE164984 | GEO

Essential Role of Sirt2 in Type I Interferon Signaling

Project description:We provide evidence for the essential role of Sirt2 in IFN mediated signaling. Our studies demonstrate that SIRT2 controls expression of key interferon stimulated genes (ISGs) that mediate important biological functions, including anti-viral and antineoplastic responses.

2015-12-31 | GSE66033 | GEO

RNA-BisSeq for YBX1 RIP in normal T24 cell

Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).

2019-07-02 | GSE133625 | GEO

Sirt2 Inhibition Reprograms T Cell Metabolism to Confer Superior Anti-Tumor Immunity

Project description:Metabolism is a key driver of T cell functions, and the switch from oxidative-phosphorylation to aerobic glycolysis is a hallmark of T cell activation. However, the mechanisms underlying the metabolic switch remain unclear. Here, we report that the Sirtuin-2 (Sirt2) deacetylase protein functions as a metabolic checkpoint that harnesses T cell effector functions and impairs anti-tumor immunity. Specifically, upregulation of Sirt2 expression in human tumor-infiltrating T lymphocytes (TILs) negatively correlates with response to Nivolumab and TIL therapy in non-small cell lung cancer. Mechanistically, Sirt2 suppresses aerobic glycolysis by deacetylating key glycolytic enzymes. Accordingly, Sirt2-deficient T cells manifest increased glycolysis, display enhanced proliferation and effector functions, and have superior anti-tumor activity. Importantly, pharmacologic inhibition of Sirt2 endows human TILs with these superior metabolic fitness and enhanced effector functions. These findings indicate Sirt2 as an actionable target for reprogramming T cell metabolism to augment a broad spectrum of cancer immunotherapies.

2020-09-16 | GSE151421 | GEO

MS analysis of STING linear ubiquitinated sites

Project description:HEK293T cells transiently transfected with plasmids encoding Flag-STING, Myc-HOIP and HA-HOIL-1L, were lysed with buffer (20 mM Na2HPO4, 20 mM NaH2PO4, 1% NP-40, 2 mM EDTA) supplemented with 1 mM DTT, 5 mM NEM, complete protease inhibitor cocktail, PhosSTOP and 0.1% SDS. The lysates were incubated with Anti-Flag M2 Affinity Gel. The bound material was eluted with 0.1 M glycine-HCl pH 2.7, separated by electrophoresis in a 10% gel, and stained with Bio-Safe Coomassie and then for mass spectrometry analysis

2023-10-26 | PXD046415 |

Sirt2 Inhibition Reprograms T Cell Metabolism to Confer Superior Anti-Tumor Immunity

Project description:Dysregulated metabolism is a key driver of maladaptive tumor-reactive T lymphocytes within the tumor microenvironment (TME). Actionable mechanisms that rescue the effector activity of anti-tumor T cells in a metabolically restricted TME remain elusive. Here, we report that the Sirtuin-2 (Sirt2) protein deacetylase functions as a master metabolic checkpoint that inhibits T cell metabolic fitness and impairs T cell effector functions and anti-tumor immunity. Mechanistically, Sirt2 suppresses glycolysis and oxidative-phosphorylation (OxPhos) by deacetylating key enzymes involved in glycolysis, tricarboxylic acid (TCA)-cycle, fatty acid oxidation (FAO) and glutaminolysis. Accordingly, Sirt2-deficient T cells exhibit a hyper-metabolic activity with increased glycolysis and OxPhos, resulting in enhanced proliferation and effector functions at tumor beds and subsequently exhibiting superior anti-tumor activity. Importantly, pharmacologic inhibition of Sirt2 endows human lung tumor-infiltrating lymphocytes (TILs) with these superior metabolic fitness and enhanced effector functions. Furthermore, upregulation of Sirt2 expression in human TILs negatively correlates with response to Nivolumab and TIL therapy in non-small cell lung cancer (NSCLC). Our findings unveil Sirt2 as an unexpected actionable target for reprogramming T cell metabolism to augment a broad spectrum of cancer immunotherapies.

2020-06-16 | PXD019763 | Pride

transcriptome analysis of SIRT2 dependent genes modulated during infection by Listeria monocytogenes

Project description:tissue culture cells were treated with an inhibitor to the deacetylase SIRT2 (AGK2) and infected with Listeria monocytogenes

2013-06-01 | E-MEXP-3912 | biostudies-arrayexpress

Metabolic control of adaptive b-cell proliferation by the protein deacetylase SIRT2

Project description:Selective and controlled expansion of endogenous b-cells has been pursued as a potential therapy for diabetes. Ideally, such therapies would preserve feedback control of b-cell proliferation to avoid excessive b-cell expansion and an increased risk of hypoglycemia. Here, we identified a regulator of b-cell proliferation whose inactivation results in controlled b-cell expansion: the protein deacetylase Sirtuin 2 (SIRT2). Sirt2 deletion in b-cells of mice increased b-cell proliferation during hyperglycemia with little effect in homeostatic conditions, indicating preservation of feedback control of b-cell mass. SIRT2 restrains proliferation of human islet b-cells cultured in glucose concentrations above the glycemic set point, demonstrating conserved SIRT2 function. Analysis of acetylated proteins in islets treated with a SIRT2 inhibitor revealed that SIRT2 deacetylates enzymes involved in oxidative phosphorylation, dampening the adaptive increase in oxygen consumption during hyperglycemia. At the transcriptomic level, Sirt2 inactivation has context-dependent effects on b-cells, with Sirt2 controlling how b-cells interpret hyperglycemia as a stress. Finally, we provide proof-of-principle that systemic administration of a GLP1-coupled Sirt2-targeting antisense oligonucleotide achieves b-cell selective Sirt2 inactivation and stimulates b-cell proliferation under hyperglycemic conditions. Overall, these studies identify a therapeutic strategy for increasing b-cell mass in diabetes without circumventing feedback control of b-cell proliferation.

2025-07-17 | GSE252859 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data