Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies.
Extractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).
Freeman
J W
JW
Chatterjee
A
A
Ross
B E
BE
Busch
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002466:Cell Nucleolus / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D009698:Nucleoproteins / Q000276:immunology*; D010750:Phosphoproteins / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009698:Nucleoproteins; D010750:Phosphoproteins; C036825:phosphoprotein C23