A distinct NF-κB–related complex binds to the proximal CD23 promoter region in pt1-LCL^tetcells. (A) Schematic representation of the proximal CD23b promoter is shown, highlighting putative transcription factor binding sites for Sp1, NF-κB, NF-AT, STAT6, and AP1. Underscoring arrows indicate the recognition sites of probes used in the following EMSAs. (B) Nuclear extracts from LCL^tet cells were incubated with probes CD23b-I (lanes 1-4), CD23b-II (lanes 5-8), and CD23b-III (lanes 9-12). Lane 8, denoted by an arrow, reveals a unique complex formation binding to probe CD23b-II in pt1 extract. (C) Further analysis of the CD23b-II probe was performed with control D11 and pt1-LCL^tet nuclear extracts. The following antibodies were added in increasing amounts to the indicated lanes: p50 (lanes 3-6), p65 (lanes 7-10), p52 (lanes 11-14), purified rabbit IgG (lanes 15-16), and c-Rel (lanes 17-20). (D) EMSA with a probe that spans the identified NF-κB binding site in the CD23a promoter (nucleotides –109 to –76). An unlabeled NF-κB competing oligonucleotide was added in 15-fold excess (lanes 3 and 11). The following supershifting antibodies were added to the indicated lanes: p50 (lanes 4 and 12), p65 (lanes 5 and 13), c-Rel (lanes 6 and 14), RelB (lanes 7 and 15), p52 (lanes 8 and 16), and purified rabbit IgG (lanes 9 and 17). Arrows indicate NF-κB–related complexes, and supershifted complexes are denoted by asterisks.