RRC ID 44403
Author Iwamoto M, Asakawa H, Ohtsuki C, Osakada H, Koujin T, Hiraoka Y, Haraguchi T.
Title Monoclonal antibodies recognize gly-leu-phe-gly repeat of nucleoporin nup98 of tetrahymena, yeasts, and humans.
Journal Monoclon Antib Immunodiagn Immunother
Abstract Nucleoporin Nup98, an essential component of the nuclear pore complex, has multifunctional roles in nuclear functions including transcriptional regulation and nucleocytoplasmic transport. These functions mostly depend on a Gly-Leu-Phe-Gly (GLFG) sequence appearing repetitively in the N-terminal region of Nup98. As the GLFG sequence is well conserved among Nup98s from a wide variety of species including humans, yeasts, and ciliates such as Tetrahymena thermophila, a specific antibody that recognizes the GLFG sequence is expected to detect various Nup98s from a wide-range of species. To generate monoclonal antibodies specific to the GLFG repeat of Nup98, we used two synthetic polypeptides derived from the macronuclear Nup98 of T. thermophila as an antigen. We obtained two monoclonal antibodies (MAbs), 13C2 and 21A10, that recognize Nup98s in indirect immunofluorescence staining and Western blot analysis of T. thermophila. Peptide array analysis of these monoclonal antibodies located the position of their epitopes at or near GLFG residues: the epitope recognized by the 13C2 MAb is FGxxN (x being any amino acid), and the epitope recognized by the 21A10 MAb is GLF. As expected by their epitopes, these monoclonal antibodies also recognize Nup98 homologs expressed by human cells and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, indicating that 13C2 and 21A10 MAbs recognize Nup98 epitopes common to phylogenetically distinct organisms. Thus, these MAbs are useful in studying a wide variety of biological phenomena that involve Nup98, ranging from ciliate nuclear dimorphism to NUP98-related human leukemia.
Volume 32(2)
Pages 81-90
Published 2013-4-1
DOI 10.1089/mab.2012.0118
PMID 23607342
PMC PMC3732132
MeSH Amino Acid Sequence Animals Antibodies, Monoclonal, Murine-Derived / chemistry* Antibody Specificity Epitope Mapping Fluorescent Antibody Technique, Indirect HeLa Cells Humans Hybridomas Mice Mice, Inbred BALB C Microscopy, Fluorescence Molecular Sequence Data Nuclear Pore Complex Proteins / chemistry Nuclear Pore Complex Proteins / immunology* Protozoan Proteins / immunology* Repetitive Sequences, Amino Acid / immunology Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins / immunology* Schizosaccharomyces Schizosaccharomyces pombe Proteins / immunology* Tetrahymena thermophila
Human and Animal Cells