FIG. 7.

PIAS1 can block the DNA binding activity of NF-κB. (A) Nuclear extracts from MCF-7 cells untreated or treated with TNF-α for 15 min were analyzed by EMSA using an oligonucleotide containing the NF-κB binding site as a probe. The identity of the shift band was examined by incubating with anti-p65, anti-p50, anti-E2F, or rabbit IgG as indicated. (B) The same extracts from panel A were mixed with increasing amounts of purified GST (lanes 3 to 5) or GST-PIAS1 proteins (lanes 6 to 8) followed by EMSA analysis. (C) The same procedure as described for panel B was carried out except that an SP-1 oligonucleotide was used as a probe. (D) Luciferase assays were performed with 293T cells with an expression construct encoding Gal4 (gray bar) or a Gal4-p65 fusion protein (black bar), increasing amounts of Flag-PIAS1, and either 5×Gal4 reporter (left) or 2×NF-κB reporter (right). Shown is the average of results from three independent experiments.