Effect of oncogenic ras expression on NF-kB in myeloma cells.

(A) Two separate experiments (left panels) in which EMSA was performed on control, wild-type ras–expressing (WT), mutant N-ras (N) or K-ras (K) cells after 48 hours of IL-6 depletion with the use of the kB-binding DNA oligonucleotide. Wild-type cells were also depleted of IL-6 for 48 hours and then re-exposed to IL-6 (100 U/mL) for 15 or 30 minutes prior to EMSA (right panel). (B) EMSAs performed on mutant K-ras or N-ras cells after 48 hours of IL-6 depletion. With K-ras nuclear extracts (left panel), cold competition was performed with 100-fold molar excess of unlabeled wild-type (cold WT) or mutant (cold MU) kB oligonucleotides. With N-ras nuclear extracts (right panel), similar cold competition experiments as well as supershift experiments were performed. Supershift experiments were performed as described in “Materials and methods” with control antibody or anti-p50 or anti-p65 antibodies. (C) The kappaB reporter expression was analyzed for wild-type (WT)– and mutant N-ras (N-ras)–expressing cells after 48 hours of IL-6 depletion. White open bars (■) are cells transfected with p5x-kB-luc, and black bars (▪) are cells transfected with a plasmid that lacked the 5 kB-binding elements. Data are shown as relative light units (RLUs), means ± SDs from 3 separate experiments after normalization for transfection efficiency. *Significantly different from corresponding wild-type group, P < .05.