MassIVE MSV000080920

Partial Public PXD006303

MudPIT analysis of proteins interacting with the retinoic acid coactivator Rere/Atrophin2 in mouse embryos

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Description

To 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. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Retinoic acid coactivator ; RA signaling ; embryonic bilateral symmetry ; Histone deacetylase

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens

Publications

Vilhais-Neto GC, Fournier M, Plassat JL, Sardiu ME, Saraf A, Garnier JM, Maruhashi M, Florens L, Washburn MP, Pourquié O.
The WHHERE coactivator complex is required for retinoic acid-dependent regulation of embryonic symmetry.
Nat Commun. 2017 Sep 28;8(1):728. Epub 2017 Sep 28.

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