Development of a two color immunofluorescence stain and immunolocalization method for N-myc and c-myc oncoproteins with a newly generated mouse IgM anti N-myc antibody.
A new mouse monoclonal antibody specific for N-myc oncoprotein was generated and used in combination with an anti-c-myc antibody to develop two color immunofluorescence staining and ultrastructural immunolocalization of N-myc and c-myc in well established (SK-N-SH; CHP 126) and in newly established neuroblastoma (NB) cell lines. Analysis and quantitation of c-myc and N-myc in dually stained cells was done by flow cytometry. Immunolocalization was done by staining with immunogold secondary antibodies and transmission electron microscopy. The results obtained from analysis of 13 newly established NB cell lines revealed, great heterogeneity in the expression of N-myc oncoprotein with 10/13 cell lines over expressing the protein. C-myc oncoprotein was also expressed in all cell lines, however, the level of expression was 4-10-fold lower than the N-myc oncoprotein. Localization studies of c-myc and N-myc oncoproteins on the level of light microscopy and electron microscopy revealed exclusive nuclear localization of c-myc whereas N-myc was localized to the nucleus and to the cytoplasm.
Gazitt
Y
Y
Department of Pediatrics Hematology Oncology, University of Florida Medical Center, Gainesville 32610.
He
Y J
YJ
Erdos
G W
GW
Chang
L
L
Ashktorab
H
H
Cohen
R J
RJ
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D006801:Humans; D007075:Immunoglobulin M; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D005810:Multigene Family; D009447:Neuroblastoma / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis*
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D016271:Proto-Oncogene Proteins c-myc; D016650:Fluorescein-5-isothiocyanate