Development of Monoclonal Antibodies Against Human IRF-5 and Their Use in Identifying the Binding of IRF-5 to Nuclear Import Proteins Karyopherin-alpha1 and -beta1.
10.3349/ymj.2008.49.6.1023
- Author:
Soo In YEON
1
;
Ju Ho YOUN
;
Mi Hwa LIM
;
Hye Ja LEE
;
Young Mok KIM
;
Ji Eun CHOI
;
Jae Myun LEE
;
Jeon Soo SHIN
Author Information
1. Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea. jsshin6203@yuhs.ac
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
Human IRF-5;
monoclonal antibody;
nuclear import protein;
karyopherins
- MeSH:
Animals;
*Antibodies, Monoclonal;
Base Sequence;
Cell Line;
Cross Reactions;
DNA Primers/genetics;
Humans;
Interferon Regulatory Factors/genetics/*immunology/*metabolism;
Mice;
Mice, Inbred BALB C;
NIH 3T3 Cells;
Protein Binding;
Recombinant Proteins/genetics/immunology/metabolism;
alpha Karyopherins/*metabolism;
beta Karyopherins/*metabolism
- From:Yonsei Medical Journal
2008;49(6):1023-1031
- CountryRepublic of Korea
- Language:English
-
Abstract:
PURPOSE: IRF-5 is a direct transducer of virus-mediated and TLR-mediated signaling pathways for the expression of cytokines and chemokines which form homodimers or heterodimers with IRF-7. However, direct IRF-5-specific monoclonal antibodies (mAbs) are not available at present. These could be used to further evaluate the functions of IRF-5. In this study, we produced and characterized three mouse mAbs to human IRF-5. The binding of IRF-5 to nuclear import proteins was first identified using a mAb. MATERIALS AND METHODS: His-tagged human IRF-5 protein spanning amino acid residues 193- 257 was used as an antigen and three mAbs were produced. The mAbs were tested with ELISA, Western blot analysis (WB), immunofluorescent staining (IF), and immunoprecipitation (IP). In addition, the nuclear import protein which carried phosphorylated IRF-5 was identified using one of these mAbs. RESULTS: MAbs 5IRF8, 5IRF10 and 5IRF24 which reacted with the recombinant His-IRF-5(193-257) protein were produced. All mAbs bound to human IRF-5, but not to IRF-3 or IRF-7. They could be used for WB, IF, and IP studies. The binding of phosphorylated IRF-5 to karyopherin-alpha1 and -beta1 was also identified. CONCLUSION: Human IRF-5-specific mAbs are produced for studying the immunologic roles related to IRF-5. Phosphorylated IRF-5 is transported to the nucleus by binding to nuclear import proteins karyopherin-alpha1 and -beta1.
