Anti-NUP98 Antibody [2H10] (A378)

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.

Nup98 is a component of nuclear pore complexes, which are large protein assemblies embedded in the nuclear envelope. Previous studies have shown that Nup98 interacts with several transport factors and plays a critical part in nuclear trafficking. However, the mechanism by which Nup98 contributes to nuclear trafficking is not clear. The present study reports on the preparation of a monoclonal antibody (MAb) directed against human Nup98. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. This antibody, MAb 2H10, specifically recognized Nup98, as evidenced by immunoblotting using a nuclear membrane fraction. In immunostaining using MAb 2H10, a punctuate nuclear rim staining pattern was observed. This MAb will be useful in immunoblotting and immunolocalization experiments in various cells and tissues, as well as further analyses of the biological function and cellular dynamics of this protein.