Spontaneous activation of NF-κB in HMC-1 cells. (A) NF-κB binding activity in nuclear extract obtained from HMC-1 cells was detected through EMSA (lane 1). Unlabeled consensus oligonucleotides (lane 2), but not mutant oligonucleotides (lane 3), competed the positive reaction. Antibodies against the p65 subunit (lane 4) and the p50 subunit (lane 6) of NF-κB shifted an electric mobility of the band to the upper position. On the other hand, anti-cRel antibody did not alter the mobility (lane 5). (B) Phosphorylation of IκBα in HMC-1 cells was detected by Western blotting (lane 1, HeLa cells; lane 2, HeLa cells stimulated with TNF-α; lane 3, HMC-1 cells).