Generation and characterization of a monoclonal antibody against NPI-1 subfamily of importin alpha.
Most karyophilic proteins are transported into the nucleus through the importin-mediated pathway. Importin alpha acts as a receptor for classical nuclear localization signal (NLS)-containing proteins. At present, the existence of several isoforms of importin alpha in mammals is known. In this study we report on the generation of a rat monoclonal antibody (MAb) 2D9 to importin alpha NPI-1 subfamily members (importin alpha5/NPI-1 and importin alpha7/S2) using the rat lymph node method and the characterization of this antibody. In several different cultured cell extracts, MAb 2D9 reacted to endogenous NPI-1 subfamily in Western blotting experiments. Epitope mapping using recombinant deletion mutants indicated that MAb 2D9 recognized arm motif in importin alpha5/NPI-1. Using immunofluorescence, MAb 2D9 detected NPI-1 subfamily in the cytoplasm of HeLa cells. Moreover, endogenous NPI-1 subfamily was dominantly localized in the nuclei of H(2)O(2)-treated HeLa cells, suggesting that NPI-1 subfamily accumulates in the nucleus in response to oxidative stress, like importin alpha1/Rch1.
Tachibana
Taro
T
Department of Bioengineering, Graduate School of Engineering, Osaka City University, Osaka, Japan. tarou@bioa.eng.osaka-cu.ac.jp
Sakaguchi
Naoko
N
Miyamoto
Yoichi
Y
Sekimoto
Toshihiro
T
Yoneda
Yoshihiro
Y
Azuma
Masayuki
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D000918:Antibody Specificity; D019556:COS Cells; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007118:Immunoassay / Q000379:methods; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D028901:alpha Karyopherins / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; C423142:KPNA1 protein, human; D011994:Recombinant Proteins; D028901:alpha Karyopherins; D006861:Hydrogen Peroxide