EMSA targeting NF-κB sites. (A) Nuclear extracts from RAW264.7 cells stimulated with RANKL (50 ng/mL) for 15 minutes were analyzed by EMSA. Gel shift assays were performed using oligonucleotides corresponding to the COX-2 distal NF-κB site (nucleotides –457/–429) and the COX-2 proximal site (nucleotides –233/–206). The COX-2 distal site (nucleotides –457/–429) probe containing a mutation in NF-κB element abolished the complex formation. (B) As described in panel A, except that nuclear extracts were prepared from RAW-RacN17 cells. Gel shift assays were performed using oligonucleotides corresponding to the COX-2 distal NF-κB site (nucleotides –457/–429). (C) Gel shift assays were performed using oligonucleotides containing the 2 tandemly repeated NF-κB sites derived from the HIV long terminal repeat. NS indicates a nonspecific protein complex. The arrows point to free probes. Similar results were obtained in 2 additional experiments.