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Identification of binding proteins of the Xenopus morphogenetic factor, Tumorhead
Author(s) -
AliceaTorres Kevin M.,
Lopez Edgardo,
Flores Noelia,
Vega Irving E.,
Traverso Edwin E.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1035.3
Subject(s) - xenopus , immunoprecipitation , microbiology and biotechnology , biology , phenotype , proteomics , mutant , epitope , proteome , inner cell mass , embryo , tandem mass spectrometry , chemistry , embryogenesis , biochemistry , mass spectrometry , genetics , cell culture , gene , antibody , blastocyst , chromatography
Tumorhead (TH) is a protein that regulates cell proliferation during early embryogenesis in Xenopus laevis. To understand how TH functions at the molecular level, we have taken a mass spectrometry‐based proteomics approach to identify TH interacting proteins. TH immunoprecipitates (IPs) were prepared from cell extracts derived from Xenopus oocytes and embryos previously injected with mRNAs encoding epitope‐tagged versions of either wild type TH, or a membrane‐bound mutant of TH, which might serve as a more efficient bait. The TH IPs were subjected to polyacrylamide gel electrophoresis, followed by silver staining to visualize the various proteins that are present in the IPs. Protein bands present in TH IPs but absent in samples generated from control‐injected embryos are being processed for nanoelectrospray ionization tandem mass spectrometry to determine their identity. Once these proteins have been identified, we will perform traditional co‐immunoprecipitation and co‐localization experiments to confirm their interaction with TH. We will also perform gain and loss of function of the identified proteins to determine if any resulting phenotype phenocopies the corresponding TH gain and loss of function phenotypes. These experiments will allow us to start addressing if the interaction between TH and the identified proteins is important for TH's role in the control of cell proliferation.