Diversity of cellular molecules in human cells detected by monoclonal antibodies reactive with c-myc proteins produced in Escherichia coli.
Six clones of monoclonal antibodies, MYC-1 to -6, were prepared by using two kinds of truncated c-myc proteins, p23 and p42, produced in Escherichia coli as immunogens. Analysis with enzyme-linked immunosorbent assays and immunoblotting assays with peptides produced in Escherichia coli showed that 5 clones of monoclonal antibodies, MYC-1 to -4 and -6, were reactive with c-myc protein encoded by exon 2. The remaining one clone, MYC-5, was reactive with the portion of c-myc protein encoded by exon 3. All monoclonal antibodies were also reactive with phosphorylated c-myc protein produced by insect cells infected by the baculovirus expression vector with the human c-myc gene. With immunoblotting assays using cellular lysates, MYC-1 and -3 detected bands at the levels of 58 kDa and 60 kDa, MYC-5 detected a band at 56 kDa and MYC-6 detected bands at 68 kDa and 75 kDa. All of these bands were detectable in nuclear extracts of HL-60 and Colo320, both of which have amplified c-myc genes, and also the extract of RmycYl which is the c-myc gene transfectant into 3Yl rat cells. None of them was detectable in peripheral blood mononuclear cells and 3Yl, both of which lacked activated c-myc genes. This indicates that these nuclear proteins are either c-myc gene products or molecules closely related to the c-myc gene. The remaining two clones, MYC-2 and -4, detected a band at the level of 85 kDa in cytoplasmic extracts of all the above-mentioned cells independent of the presence of the c-myc gene. This suggests that 85 kDa protein might be irrelevant to the c-myc gene. The 56 kDa protein was detectable by MYC-5 in phytohemagglutinin-stimulated peripheral blood mononuclear cells as well as leukemic cells of some patients.
Naoe
T
T
Department of Internal Medicine, Nagoya University Branch Hospital.
Nozaki
N
N
Yamada
K
K
Okazaki
T
T
Nakayama
E
E
Kurosawa
Y
Y
Shiku
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains / Q000276:immunology; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D016271:Proto-Oncogene Proteins c-myc; D011519:Proto-Oncogenes; D014407:Tumor Cells, Cultured / Q000032:analysis
D000911:Antibodies, Monoclonal; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D011505:Protein-Tyrosine Kinases