Fig. 5. ß₃-endonexin binds to p50/p65 complex and inhibits p50-DNA binding. (A) Immunoprecipitation. Confluent monolayers of endothelial cells were transfected with GFP/En-S, GFP/En-L or control vector. Immunoprecipitation was performed with anti-p65 or anti-GFP mAb and Agarose A. Thereafter, immunoprecipitate was probed by anti-GFP or anti-p65, by immunoblotting. (B) Pull-down. Recombinant p50 was incubated with GST/En-S, GST/En-L or GST, which were immobilized on Sepharose. The precipitate and the supernatant were probed by immunoblotting using anti-p50 mAb. Anti-GST-blotting showed equal expression of the fusion proteins. (C) EMSA. Recombinant p50 was incubated with increasing ratios (1:2, 1:3, 1:4) of GST-En-S, GST-En-L or GST, and binding of B-DNA was determined by EMSA. (D) Pulldown. Recombinant p50 was incubated with double-stranded consensus or mutated B oligonucleotides and subsequently precipitated with immobilized GST/En-S or GST/En-L proteins. The precipitate was evaluated for p50 binding by immunoblotting. Equal amounts of GST proteins was confirmed with an anti-GST pAb. (E) Pull-down. As described in D, p50 protein was incubated with double-stranded B consensus or B uPAR oligonucleotides, which contained the specific NF-B sequence of the uPAR promoter and GST/En-L. B uPAR was used in raising ratios.

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