Monoclonal antibodies recognizing different epitopes of the 27-kDa gap junction protein from rat liver.
Monoclonal antibodies (2-3E2, 6-3G11, and 7-3H6) against gap junction plaques purified from rat liver were prepared and characterized. Immunoblot analysis of liver gap junctions revealed that all three antibodies reacted with the 27-kDa protein, but not with the 22-kDa one. The 2-3E2 and 6-3G11 antibodies both reacted with the 27-kDa protein in gap junctions purified from livers of the rat, mouse, rabbit, and guinea pig; the 7-3H6 antibody, however, failed to react with the 27-kDa protein from guinea pig liver. The 7-3H6 antibody reacted strongly with the 24- to 26-kDa degradation products of the 27-kDa protein. Indirect immunofluorescence showed that the 6-3G11 and 7-3H6 antibodies both gave the same specific fluorescence labeling on rat liver cryosections, suggesting that these two antibodies recognized the cytoplasmic sites of the 27-kDa protein. Immunoblot analysis of protease-digested fragments from the 27-kDa protein revealed that the 7-3H6 antibody reacted with the 24- and 17-kDa fragments (including portions of the carboxyl-terminal domain of the 27-kDa protein) produced with endoproteinases Arg-C and Lys-C, respectively. Immunoblot analysis of CNBr fragments of the 27-kDa protein revealed that all three antibodies reacted with the 10-kDa fragment, which is thought to be the carboxyl-terminal domain of the 27-kDa protein. These results demonstrate that three monoclonal antibodies recognize different epitopes of the cytoplasmic sites (probably the carboxyl-terminal domain) of the 27-kDa liver gap junction protein.
Takeda
A
A
Department of Microbiology, School of Medicine, Ehime University.
Kanoh
M
M
Shimazu
T
T
Takeuchi
N
N
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D017630:Connexins; D000939:Epitopes; D015151:Immunoblotting; D008099:Liver / Q000276:immunology* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D008854:Microscopy, Electron; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D017630:Connexins; D000939:Epitopes; D008565:Membrane Proteins