Biochemical and Biophysical Research Communications
Identification and characterization of p100HB, a new mutant form of p100/NF-κB2
Section snippets
Materials and methods
Cells. Human peripheral blood lymphocytes (PBL) were isolated from healthy donors as described in [14]. HPB-ALL, Jurkat, Daudi, KG1, HUT 78, and RPMI 8402 cell lines were cultured in RPMI 1640 (Gibco-BRL) or IMDM for KG1 cells (Gibco-BRL) containing glutamine, 10% serum, and antibiotics. The HPB-ALL p100-stable clones were generated by electroporation with the pcDNA3-p100 expression vector and cultured in the presence of 1 mg/ml G418.
Electrophoretic mobility shift assay. Electrophoretic mobility
p100HB is a C-terminally truncated p100 produced by several tumor cell lines
First, we compared the production of p100 in normal peripheral blood lymphocytes, in HPB-ALL cells, and in a series of other human tumor cell lines, by Western blot. Using a monoclonal antibody specific for the p52, normal p100 was detected in human PBLs and in Jurkat, Daudi, KG1, and RPMI 8402 cell lines, but not in HPB-ALL cells (Fig. 1). In this cell line, only a faster migrating doublet band was detected, even after PMA treatment (Fig. 1, lanes 1, 9, and 10). This observation suggested that
Acknowledgements
We are grateful to Dr. Luis-Alfonso Martinez for critically reading the manuscript. This work was supported by grants from L’Association pour la Recherche contre le Cancer (ARC), La Ligue contre le Cancer, and GEFLUC to M.K. and fellowships to E.D. and V.B. from ARC.
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