A monoclonal antibody against insect CALNUC recognizes the prooncoprotein EWS specifically in mammalian cells. Immunohistochemical and biochemical studies of the antigen in rat tissues.
A monoclonal antibody against insect CALNUC was shown to recognize an 85-kDa nuclear protein specifically in mammalian cells. Amino acid sequencing of the protein purified from rat liver revealed it to be EWS, a prooncoprotein for Ewing sarcomas and related tumors. Using the antibody, distribution of EWS was studied in rat tissues fixed with 4% paraformaldehyde by immunohistochemical methods. On thaw-fixed cryosections or those of perfusion-fixed tissues, almost all cell nuclei showed the specific staining. In immersion-fixed tissues, the staining unexpectedly disappeared in particular tissues (kidney cortex, liver, etc.), although it was recovered by autoclaving the cryosections. Western blotting also demonstrated the ubiquitous expression of EWS in the tissues. In extracts from the liver, the 85-kDa band rapidly disappeared in a Ca(2+)-dependent manner, but never in the testis. The antigen was very labile in kidney homogenates even without Ca2+. Biochemical studies with digoxigenin-labeled EWS showed that the Ca(2+)-dependent disappearance was associated with upward mobility shifts of EWS. These suggested that EWS was ubiquitously expressed in rat tissues, and that the antigen was masked in particular tissues during the immersion fixation.
Kawano
J
J
Department of Anatomy, Miyazaki Medical Collage, Miyazaki 889-1692, Japan. jkawano@post.miyazaki-med.ac.jp
Nakayama
T
T
Takami
Y
Y
Kotani
T
T
Sawaguchi
A
A
Nagaike
R
R
Oinuma
T
T
Suganuma
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity / Q000187:drug effects*; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins / Q000276:immunology*; D000939:Epitopes / Q000032:analysis / Q000187:drug effects; D005557:Formaldehyde / Q000494:pharmacology; D006133:Growth Substances / Q000276:immunology*; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007150:Immunohistochemistry; D019476:Insect Proteins / Q000276:immunology; D008297:Male; D008322:Mammals; D051379:Mice; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D009928:Organ Specificity; D034802:RNA-Binding Protein EWS; D051381:Rats; D017208:Rats, Wistar; D012261:Ribonucleoproteins / Q000032:analysis* / Q000276:immunology; D014018:Tissue Distribution; D016707:Tissue Fixation / Q000379:methods; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D000939:Epitopes; D006133:Growth Substances; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D019476:Insect Proteins; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D034802:RNA-Binding Protein EWS; D012261:Ribonucleoproteins; D005557:Formaldehyde; D002118:Calcium