Diversity of cellular molecules in human cells detected by monoclonal antibodies reactive with c-myc proteins produced in Escherichia coli |
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Authors: | T Naoe N Nozaki K Yamada T Okazaki E Nakayama Y Kurosawa H Shiku |
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Affiliation: | Department of Internal Medicine, Nagoya University Branch Hospital. |
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Abstract: | 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. |
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