Production and characterization of monoclonal antibodies against amphiphysins.
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.
Jin
Y
Y
Department of Biochemistry, College of Medicine, Medical Research Institute, Chungbuk National University, Cheongju, Korea.
Kim
K Y
KY
Soung
N K
NK
Shin
E Y
EY
Kim
E G
EG
Kim
S R
SR
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000276:immunology*; D016716:PC12 Cells; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018909:src Homology Domains
D000911:Antibodies, Monoclonal; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C075637:amphiphysin; D005982:Glutathione Transferase