Exp Mol Med.  2001 Jun;33(2):69-75.

Production and characterization of monoclonal antibodies against amphiphysins

• Jin YL
• Kim KY
• Soung NK
• Shin EY
• Kim EG
• Kim SR

Abstract

Amphiphysin I and II, proteins enriched in nerve terminals, form heterodimers and interact with dynamin and synaptojanin through their Src homology 3 (SH3) domain. In order to study the expression profile of Amphs in cells and tissues and the interaction state with other cellular molecules, we have prepared specific monoclonal antibodies (mAbs) designed to bait N-terminus, middle part, and C-terminus domains of Amph I, respectively by immunizing with the expressed smaller domain molecules using the GST gene fusion system. The expression of Amphs was found to be most abundant in PC12 cells, followed by B103 cells and vascular smooth muscle cells. Western blot analysis showed a relatively high level expression of Amphs that were found in both mouse and rat brain. There appeared to be some species difference in the expression pattern, i.e. Amphs are present more in the testis than in the lungs in rats, however, they are reversed in mice. Characterization of the mAbs revealed that clone 14-23 precipitated Amph I and II, whereas clone 8-2 could only precipitate Amph I. In addition, clathrin and dynamin in a complex with Amph were captured in the precipitate formed by mAbs and identified by the Western blot analysis. Cellular distribution of Amph was visualized with confocal immunofluorescence microscopy performed using the labeled-mAbs. Taken together, these results demonstrated that mAbs provided an excellent measure for studying Amphs' expression profile and their interacting proteins.

Keyword

amphiphysin; monoclonal antibody

MeSH Terms

Animal
*Antibodies, Monoclonal
Blotting, Western
Brain/metabolism
Cells, Cultured
Dimerization
Enzyme-Linked Immunosorbent Assay
Glutathione Transferase/metabolism
Human
Mice
Mice, Inbred BALB C
Microscopy, Confocal
Nerve Tissue Proteins/*chemistry/*immunology
PC12 Cells
Precipitin Tests
Protein Binding
Protein Structure, Tertiary
Rats
Recombinant Fusion Proteins/metabolism
Support, Non-U.S. Gov't
src Homology Domains