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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.

 Soo-In Yeon  ;  Ju Ho Youn  ;  Mi Hwa Lim  ;  Hye Ja Lee  ;  Young Mok Kim  ;  Ji Eun Choi  ;  Jae Myun Lee  ;  Jeon-Soo Shin 
 YONSEI MEDICAL JOURNAL, Vol.49(6) : 1023-1031, 2008 
Journal Title
Issue Date
Animals ; Antibodies, Monoclonal* ; Base Sequence ; Cell Line ; Cross Reactions ; DNA Primers/genetics ; Humans ; Interferon Regulatory Factors/genetics ; Interferon Regulatory Factors/immunology* ; Interferon Regulatory Factors/metabolism* ; Mice ; Mice, Inbred BALB C ; NIH 3T3 Cells ; Protein Binding ; Recombinant Proteins/genetics ; Recombinant Proteins/immunology ; Recombinant Proteins/metabolism ; alpha Karyopherins/metabolism* ; beta Karyopherins/metabolism*
Human IRF-5 ; monoclonal antibody ; nuclear import protein ; karyopherins
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.
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1. College of Medicine (의과대학) > Dept. of Microbiology (미생물학교실) > 1. Journal Papers
Yonsei Authors
Shin, Jeon Soo(신전수) ORCID logo https://orcid.org/0000-0002-8294-3234
Lee, Jae Myun(이재면) ORCID logo https://orcid.org/0000-0002-5273-3113
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