Browse
Submit Data
Databases
API
Help

Dataset Information

299 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

P65-HaloTag copurifying proteins in human 293T cells

ABSTRACT: In effort to develop methodology for targeted top down mass spectrometry of NF kappa B p65 from human cells, we evaluated the utility of HaloTag for purification and analysis of recombinant protein. During our study, two datasets of bottom up LC-MS/MS were generated: one from in-gel digestion of the predominant band following p65-HaloTag purification, another from in-solution digestion of all the proteins present in a p65-HaloTag purification. p65-HaloTag copurifying proteins identified in our datasets include the known interactors c-Rel, NF-kappaB p105, NF-kappaB p100, and NF-kappaB inhibitor beta. Over 100 proteins were identified by at least two peptides using a Mascot ion cut-off score of 30.

INSTRUMENT(S): LTQ FT, LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: John Savaryn

LAB HEAD: Neil Kelleher

PROVIDER: PXD001932 | Pride | 2018-10-19

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry.

Savaryn John Paul JP Skinner Owen S OS Fornelli Luca L Fellers Ryan T RT Compton Philip D PD Terhune Scott S SS Abecassis Mike M MM Kelleher Neil L NL

Journal of proteomics 20150504

Measuring post-translational modifications on transcription factors by targeted mass spectrometry is hampered by low protein abundance and inefficient isolation. Here, we utilized HaloTag technology to overcome these limitations and evaluate various top down mass spectrometry approaches for measuring NF-κB p65 proteoforms isolated from human cells. We show isotopic resolution of N-terminally acetylated p65 and determined it is the most abundant proteoform expressed following transfection in 293T ...[more]

PMID: 25952688

Similar Datasets

Mouse livers lacking NF-kappaB RelA (p65) during bacteremia

Project description:Mouse livers lacking NF-kappaB RelA (p65) during bacteremia

| PRJNA522786 | ENA

PU.1 and C/EBPalpha synergistically program distinct response to NF-kappaB activation through establishing monocyte specific enhancers (expression data)

Project description:Unraveling the complexity of transcriptional programs coded by different cell types has been one of the central goals of cell biology. Using genome-wide location analysis, we examined how two different cell types generate different responses to the NF-kappaB signaling pathway. We showed that, after tumor necrosis factor-alpha (TNF-alpha) treatment, NF-kappaB p65 subunit binds to distinct genome locations and subsequently induces different subsets of genes in human monocytic THP-1 cells versus HeLa cells . Interestingly, the differential p65 binding in two cell types correlates with pre-existing cell-type specific enhancers prior to TNF-alpha stimulation, marked by histone modifications. We also found that two transcription factors, PU.1 and C/EBPalpha, appear to synergistically mediate enhancer creation and affect NF-kappaB target selection in THP-1 cells. In HeLa cells, co-expression of PU.1 and C/EBPalpha conferred TNF-alpha responsiveness to a subset of THP-1 specific NF-kappaB target genes. These results suggest that the diversity of transcriptional programs in mammalian cells arises, at least in part, from pre-existing enhancers that are established by cell specific transcription factors. We used Affymetrix microarray (GPL570) to obtain gene expression data for THP1 and HeLa cells before and after TNF-alpha treatment.

2011-03-15 | E-GEOD-26868 | biostudies-arrayexpress

Expression data from WT or p65-/- mouse embryonic fibroblasts (MEFs) infected with Toxoplasma gondii.

2011-01-10 | GSE25469 | GEO

Expression data from WT or p65-/- mouse embryonic fibroblasts (MEFs) infected with Toxoplasma gondii.

Project description:Toxoplasma strains have been shown to modulate host cell transcription. We have found a type II Toxoplasma gene, GRA15, which activates the nuclear translocation of the NF-kappaB p65 transcription factor. We used microarrays to determine how GRA15II modulates host cell transcription, and whether this transcription is dependent on the p65 transcription factor. WT or p65-/- MEFs were infected with type I (RH), type I GRA15II (RH GRA15II), type II (Pru), or type II GRA15KO (Pru GRA15KO) parasites for 18-24 hours. Total RNA was isolated and hybridized to Affymetrix GeneChip Human Genome U133A 2.0 arrays.

2011-01-10 | E-GEOD-25469 | biostudies-arrayexpress

Monocyte vs Macrophage Study

Project description:Human intestinal macrophages contribute to tissue homeostasis in noninflamed mucosa through profound down-regulation of pro-inflammatory cytokine release. Here, we show that this down-regulation extends to Toll-like receptor (TLR)-induced cytokine release, as intestinal macrophages expressed TLR3-TLR9 but did not release cytokines in response to TLR-specific ligands. Likely contributing to this unique functional profile, intestinal macrophages expressed markedly down-regulated adapter proteins MyD88 and Toll interleukin receptor 1 domain-containing adapter-inducing interferon beta, which together mediate all TLR MyD88-dependent and -independent NF-kappaB signaling, did not phosphorylate NF-kappaB p65 or Smad-induced IkappaBalpha, and did not translocate NF-kappaB into the nucleus. Importantly, transforming growth factor-beta released from intestinal extracellular matrix (stroma) induced identical down-regulation in the NF-kappaB signaling and function of blood monocytes, the exclusive source of intestinal macrophages. Our findings implicate stromal transforming growth factor-beta-induced dysregulation of NF-kappaB proteins and Smad signaling in the differentiation of pro-inflammatory blood monocytes into noninflammatory intestinal macrophages. Comparison of unstimulated monocytes and macrophages, and flagellin stimulated monocytes and macrophages.

2010-07-07 | E-GEOD-22373 | biostudies-arrayexpress

PU.1 and C/EBPalpha synergistically program distinct response to NF-kappaB activation through establishing monocyte specific enhancers (expression data)

2011-03-15 | GSE26868 | GEO

PU.1 and C/EBPalpha synergistically program distinct response to NF-kappaB activation through establishing monocyte-specific enhancers (ChIP-chip data)

2011-03-15 | GSE26988 | GEO

Monocyte vs Macrophage Study

2010-06-22 | GSE22373 | GEO

Expression data from Human foreskin fibroblasts (HFFs) infected with Toxoplasma gondii.

2011-01-10 | GSE25468 | GEO

Trap-80-dependence of TNF-alpha-induced genes

Project description:In fibroblasts, p65-dependent genes can be sub-divided, depending on whether they are Trap-80-dependent or -independent. To examine the generality of this grouping, we performed a microarray analysis of wild-type and Trap-80 knock-down fibroblasts, before and after stimulation of NF-kappaB activity using TNF-alpha.

2009-04-01 | GSE12697 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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