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RPE-1 and RPE-1 trisomic AHA p-c and steady state data

ABSTRACT: We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. In addition, steady state protein levels comparing the RPE- and RPE-1 trisomic cells by standard SILAC labeling was also acquired. These datasets contain data for the human RPE-1 and RPE-1 trisomic cell lines.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Erik McShane

LAB HEAD: Matthias Selbach

PROVIDER: PXD004915 | Pride | 2016-10-06

REPOSITORIES: pride

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			Action	DRS
	Gladys_20150902_SEM_chrom_012_125mM.raw	Raw
	Gladys_20150902_SEM_chrom_012_500mM.raw	Raw
	Gladys_20150902_SEM_chrom_048_125mM.raw	Raw
	Gladys_20150902_SEM_chrom_048_500mM.raw	Raw
	Gladys_20150904_SEM_RPE1_2h_125mM.raw	Raw

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Publications

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.

McShane Erik E Sin Celine C Zauber Henrik H Wells Jonathan N JN Donnelly Neysan N Wang Xi X Hou Jingyi J Chen Wei W Storchova Zuzana Z Marsh Joseph A JA Valleriani Angelo A Selbach Matthias M

Cell 20161006 3

Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) ...[more]

PMID: 27720452

Similar Datasets

RPE-1 and RPE-1 trisomic AHA p-c and steady state data

Project description:We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. In addition, steady state protein levels comparing the RPE- and RPE-1 trisomic cells by standard SILAC labeling was also acquired. These datasets contain data for the human RPE-1 and RPE-1 trisomic cell lines.

2017-03-31 | MSV000080831 | MassIVE

NIH 3T3 AHA p-c, SILAC p-c, inhibitor, and enrichment control experiments

Project description:We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. These experiments were performed with 0, 1, 2, 4, 8, 16 and 13 h of chase. Data from 3 biological replicates are provided. Each replicate contains data from 3 experiments (0, 1, 2 and 0, 4, 8 and 0, 16, 32 hours chase) in where the 0 h time point is always heavy labelled followed by the medium and light labelled time points. In addition, AHA p-c experiments were performed using only 0, 4 and 8 h chase times in combination with different inhibitor combinations (MG132, Actinomycin D and Wortmanninin + Bafilomycin A1) or control (DMSO). Also, a control enrichment data set was created. Here heavy SILAC labelled cells were pulsed with AHA before being mixed with light cells that were not pulsed with AHA. This was followed by the click reaction and enrichment of AHA containing proteins. Label-swap experiments were also performed. Finally, a SILAC p-c data set is provided. Here light cells were pulsed with heavy (Arg10, Lys8) amino acids and then split in two. One half of the cells was chased in medium (Arg6, Lys4) amino acids and the other part was directly frozen. Label-swap experiments and experiments using different pulse and chase times were also performed. All provided datasets are from mouse fibroblast cells (NIH 3T3).

2016-10-06 | PXD004929 | Pride

Profiling of chromatin deposition bias in RPE-1 cells using Double-click-seq

Project description:We used double-click-seq method to profile chromatin deposition bias in RPE-1 cells. Untreated wild-type, p53 knock-out and several treatments (hydroxyurea, ATR inhibitor, curaxin) and their combinations were profiled for characterizing assymetry during DNA replication.

2021-10-04 | E-MTAB-8624 | biostudies-arrayexpress

RPE Redifferentiation (miRNA)

Project description:Retinal Pigment Epithelial (RPE) cells are located behind the retina and are critical for photoreceptor survival. Loss of RPE is associated with several pathogenic conditions such as Age Related Macular Degeneration and Retinitis Pigmentosa. RPE derived from human embryonic stem cells (hESC) offer a potential source for producing these cells for therapy. Here we report the molecular and cellular characterization of RPE differentiated from hESC. hESC derived RPE are capable of proliferation and lose their epithelial characteristics before becoming confluent and re-differentiating back into their typical pigmented, cobblestoned appearance. During the proliferative phase, they adopt a mesenchymal morphology and express mesenchymal markers. Our results demonstrate that this apparent Epithelial-Mesenchymal Transition is not regulated by the classical EMT transcription factors SNAIL and SLUG. Furthermore, it is possible to regulate RPE de-differentiation and re-differentiation by modulating the Wnt and BMP pathway respectively. These findings further our understanding of the genesis and expansion of RPE which is essential for their therapeutic use.

2013-01-17 | E-MTAB-960 | biostudies-arrayexpress

The interactome of the clathrin adaptor AP2 is modulated by the phosphorylation status of its µ2 subunit.

Project description:The clathrin adaptor AP2 is phosphorylated at a single site (Thr156) of its µ2 subunit. To gain insight how phosphorylation influences the interactome of AP2, we used human RPE cells which that were treated with LP-935509, an inhibitor of the adaptor-associated kinase AAK1. Kinase inhibition and loss of µ2 phosphorylation was verified with phospho-specific antibodies in westernblots of total cell extracts. We then used SILAC labelled cells and immune-isolated AP-2 and subjected bound proteins to mass spectrometry to identify all bound proteins and to determine phosphorylation-dependent changes in the interactome.

2019-11-12 | PXD013468 | Pride

MicroRNAs of the RPE are essential for RPE differentiation and photoreceptor maturation (mRNA)

Project description:Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPEs and important for their development and maintenance, virtually nothing is known about the in vivo roles of non-protein coding transcripts in RPEs. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and few were implicated to play role in RPE based on studies in cell lines. Herein, through RPE specific conditional mutagenesis of Dicer1 or DGCR8, the importance of miRNA for RPE differentiation was uncovered. Interestingly, miRNAs were found to be dispensable for maintaining the RPE fate and survival, and yet they are essential for acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly miRNAs of the RPE were found to be required for maturation of the adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The profiles of miRNA and mRNA altered in the Dicer1 deficient RPE point to a key role of miR-204 in regulation of RPE differentiation program in vivo and uncovers the importance of additional novel RPE miRNAs. The study exposes the combined regulatory activity of miRNAs of the RPE, which is required for RPE differentiation and for the development of the adjacent neuroretina. Effect of Dicer 1 deficiency on RPE miRNA and mRNA.

2015-06-16 | E-GEOD-69881 | biostudies-arrayexpress

MicroRNAs of the RPE are essential for RPE differentiation and photoreceptor maturation (microRNA)

2015-06-16 | E-GEOD-69882 | biostudies-arrayexpress

Development and evaluation of a SILAC method for proteomic analysis of rat primary microglia

Project description:Microglia play important and dynamic roles in mediating a variety of physiological and pathological processes during the development, normal function, and degeneration of the central nervous system. We report here the development of a SILAC-labeled model system suitable for mass spectrometry-based proteomic profiling in primary rat microglia. A mixed primary glia culture was first established by isolating newborn brain cells from Sprague-Dawley rats and maintaining the culture in a fetal bovine serum-supplemented and chemically defined media containing either the regular amino acids (light label media) or stable isotope-labeled amino acids (heavy label media). After isolation of microglia from the mixed glia cultures, mass spectrometric analysis indicated that the incorporation efficiency of the heavy-labeled amino acids was between 70-80%. Upon stimulation with bacterial endotoxin lipopolysaccharide to induce classical (M1) activation, the light- and heavy-labeled primary microglia responded indistinguishably as judged by the up-regulation of inducible nitric oxide synthase. More important, the relative quantitation accuracy and proteome coverage obtained by the reported SILAC model allowed for identification of classical microglial activation markers and pathways involved in the proinflammatory response of microglia as determined by bioinformatic analysis. The establishment of this viable primary microglia SILAC model will further expand our capacity for global scale proteomic profiling of pathways mediating the multifaceted microglial responses in various physiological and pathological processes.

2016-04-07 | PXD002759 | Pride

Expresion data from primary retinal pigment epithelium (RPE) and immortalized RPE

Project description:We used microarrays to detail the global gene expression of primary RPE and immortalized RPE. We investigated highly expression genes in immortalized RPE.

2017-09-07 | GSE80985 | GEO

H4K20me2 SILAC Nucleosome Affinity Purification

Project description:Goal of this project was the identification of chromatin interacting proteins whose binding is differentially regulated by di-methylation of lysine 20 on histone H4 (H4K20me2). To achieve this unodified and H4K20me2-modified histone H4 were generated by native chemical ligation and assembled into recombinant di-nucleosomes. The di-nucleosomes were immobilized on streptavidin-coated beads via the biotinylated di-nucleosomal DNA and used for nucleosome affinity purifications to identify proteins regulated by H4K20me2 from SILAC-labelled HeLaS3 nuclear extracts (Arg10 and Lys8). SILAC affinity purifications were carried out in "forward" (heavy extract on modified nucleosome and light extract on unmodified nucleosome) and "reverse" (light extract on modified nucleosome and heavy extract on unmodified nucleosome) label-swap experiments and protein abundances were quantified by MaxQuant.

2019-02-27 | PXD009281 | Pride

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