Colocalization of GP125/CD98 with Tropomyosin Isoforms at the Cell-Cell Adhesion Boundary

Two monoclonal antibodies designated as 1FS and 4B10 were obtained on screening for reactivities to CD98-associated molecules by sandwich-type enzyme-linked iinmunosorbent assaying using hybridoma culture supernatants as the solid phase, cell lysates as an antigen source, and a mixture of biotinylat...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2000-02, Vol.127 (2), p.253-261
Main Authors: Shishido, Takao, Ohkawa, Mayumi, Itoh, Akihiro, Enomoto, Takemi, Hashimoto, Yoshiyuki, Masuko, Takashi
Format: Article
Language:English
Subjects:
Actins - metabolism
Animals
Antibodies, Monoclonal
Antigens, CD - immunology
Antigens, CD - metabolism
Carrier Proteins - immunology
Carrier Proteins - metabolism
Cell Adhesion - physiology
cell-cell adhesion
cytoskeleton
Female
Fusion Regulatory Protein-1
GP125/CD98
Humans
Hybridomas - immunology
Hybridomas - metabolism
Male
Mice
Mice, Inbred BALB C
Microscopy, Confocal
monoclonal antibody
Protein Isoforms - metabolism
Rabbits
Rats
Reference Values
Subcellular Fractions
tropomyosin
Tropomyosin - metabolism
Tumor Cells, Cultured
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Summary:Two monoclonal antibodies designated as 1FS and 4B10 were obtained on screening for reactivities to CD98-associated molecules by sandwich-type enzyme-linked iinmunosorbent assaying using hybridoma culture supernatants as the solid phase, cell lysates as an antigen source, and a mixture of biotinylated antibodies to CD98HC as a detector. flow cytometric analysis with microspheres in combination with iFS, 4B10, and antiCD9SHC also indicated the association of antibody-defined antigen(s) with CD9S. 1FS and 4B10, stained fibrillate components in fixed and permeated cells but were not reactive with unfixed live cells, suggesting that epitopes reside in the cytoskeleton–associated structure in the intracellular region. Two-color iminunostaining followed by confocal microscopy revealed the colocalization of the antigen with CD98 at the cell-cell adhesion boundary of HeLa cells. iFS detected proteins with relative molecular masses of 33,000 to 43,000 on immunoblotting analysis involving cell lysates of human and rat cell lines. Analysis with a purified tropomyosin specimen from rabbit skeletal muscle demonstrated that iFS and 4BiO recognize tropomyosin. Two-dimensional gel electrophoresis followed by immunoblotting analysis revealed that iFS recognizes various tropomyosin isoforms. These results indicated that CD98 physically associates directly or indirectly with tropomyosin, and that this association is closely related to the cell-cell interaction
ISSN:0021-924X
DOI:10.1093/oxfordjournals.jbchem.a022602