Comparison of the composition of NFκB DNA-binding activity present in IL-3–dependent parental H7 cells versus derivative cell lines with p210Bcr-Abl, gag-akt, RafCAAX, or both gag-akt/RafCAAX demonstrates unique presence of c-rel in IL-3–dependent H7 cells.

(A) On the left, extracts were made from cells deprived of IL-3 and then these extracts were placed into a gel mobility shift experiment with an NFκB oligonucleotide probe labeled with ³²P-ATP, and then the resulting gel was dried and autoradiographed. On the right, extracts of the cells taken from usual growth medium, including IL-3 only for H7 parental cells, H7gag-akt, and H7RafCAAX cells (no IL-3 was added to BCR-ABL.A54 or H7gag-akt/RafCAAX cells), were subjected to gel mobility shift. Arrows on left note the mobility of complexes composed of p50 homodimers, heterodimers of p65, or complexes of c-rel. (B) Extracts of cells deprived of IL-3 were placed into a gel mobility shift experiment with NFκB oligonucleotide, and the composition of complexes was established by supershift protocol using antibodies to c-rel, p65, and rel B (negative control). Exclusive appearance of c-rel was noted in H7 parental cell extracts and exclusive appearance of p65 in extracts of H7 BCR-ABL.A54 and H7gag-akt/RafCAAX cells. Supershifted complexes rose to the top of the gel (not pictured). Note that a control gel shift for constitutive complex of CREB (not shown) demonstrated that the H7gag-akt/RafCAAX extract was slightly underloaded.