FIG. 4.
In vitro analysis of a potential κB-Sp1 site at −295 to −286. (A) Time course analysis of complex binding to probe B (−306 to −278). Jurkat cells were activated with P/I for the indicated time, nuclear extracts were harvested, and EMSA analysis was performed with 32P-labeled probe B (left panel, lanes 1 to 5). Cold competition was performed with 1-h-activated Jurkat cell nuclear extracts with a 25-fold excess of consensus oligonucleotides for the transcription factors AP-1, NF-AT, wild-type probe B (W-T), or mutated probe B (Mut.) added prior to incubation with 32P-labeled probe B (lanes 7 to 10). For NF-κB and Sp1 competition, a 25-fold excess (lanes 11 and 13) and 100-fold excess (lanes 12 and 14) of consensus oligonucleotides were added to equivalent amounts of P/I-activated nuclear extracts prior to addition of 32P-labeled probe B. N.S., nonspecific binding. (B) Supershift analyses of P/I-activated Jurkat cell nuclear extracts. Nuclear extracts were subjected to EMSA analysis in the absence (lane 1) or presence of the indicated specific antisera (lanes 2 to 8), or preimmune IgG (lane 9). With two different anti-Sp1 antisera, probe B binding to the upper C2 complex was shown to be either shifted or inhibited (lanes 7 and 8). Arrows indicate the identity of the shifted complexes. (C) Mutual exclusive binding of Sp1 and NF-κB p50 to probe B. Recombinant Sp1 (4 fpu [120 ng]) was preincubated with 12,500 cpm of radiolabeled probe B in the absence (lane 2) or presence of increasing amounts (0 to 40 ng) of recombinant NF-κB p50 (lanes 3 to 6) prior to EMSA analysis. Brackets indicate the Sp1-probe B and p50-probe B complexes.