title: Phospho-Specific Antibody Probes of Intermediate Filament Proteins.
abstract: Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Accumulating data have suggested that IF protein phosphorylation dramatically changes IF structure/dynamics in cells. For the production of an antibody recognizing site-specific protein phosphorylation (a site- and phosphorylation state-specific antibody), we first employed a strategy to immunize animals with an in vitro-phosphorylated polypeptide or a phosphopeptide (corresponding to a phosphorylated residue and its surrounding sequence of amino acids), instead of a phosphorylated protein. Our established methodology not only improves the chance of obtaining a phospho-specific antibody but also has the advantage that one can predesign a targeted phosphorylation site. It is now applied to the production of an antibody recognizing other types of site-specific posttranslational modification, such as acetylation or methylation. The use of such an antibody in immunocytochemistry enables us to analyze spatiotemporal distribution of site-specific IF protein phosphorylation. The antibody is of great use to identify a protein kinase responsible for in vivo IF protein phosphorylation and to monitor intracellular kinase activities through IF protein phosphorylation. Here, we present an overview of our methodology and describe stepwise approaches for the antibody characterization. We also provide some examples of analyses for IF protein phosphorylation involved in mitosis and signal transduction.
authors.affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan; Department of Cellular Oncology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan.
authors.forename: Hidemasa
authors.initials: H
authors.lastname: Goto
authors.affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan; Department of Oncology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan.
authors.forename: Kousuke
authors.initials: K
authors.lastname: Kasahara
authors.affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan; Department of Cellular Oncology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan. Electronic address: minagaki@aichi-cc.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Inagaki
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D015335:Molecular Probes / Q000378:metabolism; D010766:Phosphorylation; D014746:Vimentin / Q000378:metabolism
chemical_list: D000906:Antibodies; D007381:Intermediate Filament Proteins; D015335:Molecular Probes; D014746:Vimentin
keywords: ELISA; Immunoblotting; Immunocytochemistry; Phosphorylation; Site- and phosphorylation state-specific antibody (phospho-specific antibody); Vimentin