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Protein‐protein interactions in fixed cells and glial tumors revealed by antibody‐based fluorescence resonance energy transfer (FRET)
Author(s) -
Brill Louis,
Eblen Scott T,
Periasamy Ammasi,
Weber Michael J,
Mandell James W
Publication year - 2006
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.20.5.a1088
Subject(s) - förster resonance energy transfer , fluorophore , epitope , transfection , microbiology and biotechnology , chemistry , immunofluorescence , antibody , biophysics , fluorescence , biology , biochemistry , immunology , physics , quantum mechanics , gene
The overall objective of our research is to quantify protein‐protein interactions in fixed pathological archival specimens. Our data demonstrate proof‐of‐principle for the use of antibody‐based fluorescence resonance energy transfer (FRET) to evaluate colocalization of components of the MEK/ERK intracellular signaling pathway, first in cultured cells and then archival tumor samples. We first validated the use of FRET in this context by measuring the well‐characterized interaction between MEK1 and ERK2 in tissue culture. Cos‐1 cells transfected with epitope‐tagged wild type or mutant (non‐MEK binding) MEK1 and ERK2 constructs were fixed in formalin and processed for indirect immunofluorescence using epitope tag antibodies and fluorophore‐conjugated secondary antibodies. FRET analysis utilizing the three filter method and the PFRET software package revealed significant FRET signal for wild type ERK2, but not the mutant. FRET between endogenous MEK1 and ERK2 was revealed in cultured U251 glioma cells, and responded to serum starvation/stimulation paradigms as predicted (low ERK1/MEK2 FRET signal in serum conditions; increased with serum starvation; see figure). Current work is aimed at analyzing FRET in archival astrocytic tumor specimens and non‐neoplastic control tissue.This work was supported by a Farrow fellowship grant from the University of Virginia Cancer Center.