Premium
Characterization of antibodies recognizing a putative extracellular epitope of the neuronal K‐Cl cotransporter, KCC2
Author(s) -
Goodier Richard J,
Williams Jeffery R,
Payne John A
Publication year - 2007
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.21.5.a532
Subject(s) - immunostaining , antiserum , epitope , antibody , polyclonal antibodies , extracellular , biology , microbiology and biotechnology , hek 293 cells , immunohistochemistry , immunochemistry , chemistry , receptor , biochemistry , immunology
Since KCC2 is an important regulator of neuronal [Cl−], its protein expression/activity helps shape GABA‐A and glycine receptor‐mediated signaling and hence influences various aspects of neuronal development, plasticity, and trauma. Recent studies indicate that physiological and pathophysiological modulation of neuronal [Cl−] may take place via changes in KCC2 surface expression. In an effort to aid future studies quantifying KCC2 surface expression, we developed rabbit polyclonal antibodies against a portion of the presumed extracellular loop of KCC2 between putative transmembrane regions 5 and 6. Antiserum collected four weeks post‐injection of a purified GST‐fusion protein (SF3) was positive for KCC2 by immunoblot. Pre‐incubation of antiserum with excess SF3 fully and specifically competed off antibody binding to KCC2 on immunoblots. Immunohistochemistry of rat cerebellar sections with purified antiserum revealed a staining pattern that was similar to that of a previously characterized specific KCC2 antibody. Furthermore, immunostaining of KCC2 expressed in HEK cells under permeabilizing conditions revealed specific cell surface staining. Our findings indicate that these new antibodies specifically recognize KCC2 and may be useful for cell surface quantification. Supported by NIH NS‐36296