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Immuno‐enrichment of the novel B‐raf target FAM129B from rat lung tissue
Author(s) -
Holdman Erin Anne,
Anderson Tanner,
Todhunter Ryan,
Riedinger John,
Muhonen Wallace,
Shabb John
Publication year - 2012
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.26.1_supplement.753.4
Subject(s) - phosphorylation , polyclonal antibodies , blot , mapk/erk pathway , microbiology and biotechnology , antibody , antiserum , trypsin , chemistry , western blot , biology , biochemistry , immunology , enzyme , gene
The protein FAM129B is a recently discovered downstream target of the B‐Raf‐MEK‐ERK signaling pathway in melanoma cells. The ERK‐dependent phosphorylation of FAM129B is essential to melanoma cell invasion. FAM129B is localized to the plasma membrane in its less phosphorylated state, but becomes cytosolic upon ERK‐dependent phosphorylation. FAM129B is also expressed in normal tissue. Here we show its expression in rat lung. We hypothesize that FAM129B's subcellular location is mediated by protein‐protein interaction and that this interaction is dependent on its phosphorylation state. Polyclonal anti‐FAM129B antisera directed against three unique peptide sequences were tested to determine their efficacies in immuno‐enriching endogenous FAM129B in rat lung. Protein A Sepharose‐coupled antibodies were incubated with rat lung lysate. Specifically bound proteins were eluted and characterized by Western Blotting and silver staining. The region of the gel corresponding to FAM129B was subjected to trypsin digestion. Peptides were analyzed by MALDI TOF/TOF mass spectrometry (MS). Spectra were searched against a rat database and matched to FAM129B to give 40% sequence coverage. FAM129B was selectively enriched with two of the three antibodies. Further studies will use MS to identify candidate interacting partners of FAM129B. Supported by North Dakota INBRE (NCRR P20 RR016471); and UND School of Medicine.