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PXD006303

PXD006303 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleMudPIT analysis of proteins interacting with the retinoic acid coactivator Rere/Atrophin2 in mouse embryos
DescriptionTo identify proteins binding to Rere, we first generated a mouse colony homozygous for both the T-Cre transgene and the RS-Rere-HA allele. Thus, all the embryos generated by crossing these mice expressed Rere-HA only in mesodermal tissues. We collected around 600 E10.5 embryos and prepared whole-cell extracts using either a low salt (350 mM NaCl) or a high salt (420 mM NaCl) extraction buffer. For the low salt condition, affinity purification was done at 150 mM NaCl; whereas for the high salt protocol, it was performed at 300 mM NaCl. Each affinity purification was performed on biological replicates of protein extracts from about 50-70 embryos prepared on different days. Two different anti-HA antibodies from Sigma and Roche were used for the affinity purification. For each of the conditions, six elutions were performed with HA peptide, and the last elution with 2 percent SDS to completely remove all the proteins attached to the beads. Samples were analyzed by MudPIT as follows: elution 1 run alone; elution 2 and 3 combined; and elutions 4 to 7 combined. A total of 11 and 12 MudPIT analyses were performed for the low and high salt conditions, respectively. The same purification strategy was applied to embryos from wild-type females to use as a negative control. Proteins were extracted in either low or high salt conditions from 108 x 2 and 100 x 2 embryos prepared on different days, then split in half and affinity purified using either the Sigma or Roche anti-HA antibodies; elutions were combined into 3 digested samples as described above and analyzed independently for a total of 12 MudPIT analyses. After TCA-precipitation, proteins were urea-denatured, reduced, alkylated, then digested with endoproteinase LysC followed by trypsin. The resulting peptide mixtures were analyzed by Multidimensional Protein Identification Technology (MudPIT). Label-free quantitative proteomics was used to identify and quantify the relative abundance of affinity-enriched proteins.
HostingRepositoryMassIVE
AnnounceDate2017-10-04
AnnouncementXMLSubmission_2017-10-04_08:49:17.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterLaurence Florens
SpeciesList scientific name: Mus musculus; common name: house mouse; NCBI TaxID: 10090;
ModificationListS-carboxamidomethyl-L-cysteine
InstrumentLTQ
Dataset History
RevisionDatetimeStatusChangeLog Entry
02017-04-13 11:25:14ID requested
12017-10-04 08:49:18announced
Publication List
Vilhais-Neto GC, Fournier M, Plassat JL, Sardiu ME, Saraf A, Garnier JM, Maruhashi M, Florens L, Washburn MP, Pourqui, é O, The WHHERE coactivator complex is required for retinoic acid-dependent regulation of embryonic symmetry. Nat Commun, 8(1):728(2017) [pubmed]
Keyword List
submitter keyword: Retinoic acid coactivator, RA signaling, embryonic bilateral symmetry, Histone deacetylase
Contact List
Laurence Florens
contact affiliationThe Stowers Institute for Medical Research
contact emaillaf@stowers.org
lab head
Laurence Florens
contact affiliationStowers Institute for Medical Research
contact emaillaf@stowers.org
dataset submitter
Full Dataset Link List
MassIVE dataset URI
Dataset FTP location
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