Fig. 1.

EGF induces formation of the c-Jun/ARNT/Sp1 complex and its binding to the Sp1 sites of the gene promoter. Cells were starved for 18 h in serum-free culture medium and then treated with 50 ng/ml EGF for a different time period as indicated. Nuclear extracts were used for the following experiments. a The Sp1, ARNT and c-Jun proteins were detected by anti-Sp1, anti-ARNT and anti-c-Jun antibodies, respectively. b–d Nuclear extracts were immunoprecipitated (IP) with antibodies (Ab) against Sp1, ARNT and c-Jun. The proteins were subjected to SDS–PAGE and analyzed by western blotting with antibodies against c-Jun, ARNT and Sp1. IgG was for the negative control of antibodies. e After EGF treatment for different time period as indicated, nuclear extracts were prepared, chromatin immunoprecipitation (ChIP, lower panel) and DNA affinity precipitation assay (upper panel) were performed. Binding of Sp1, ARNT and c-Jun to Sp1 binding sequence of 12(S)-lipoxygenase promoter (12-LOX-Sp1) was analyzed by western blot. The streptavidin-agarose beads were used as a nonspecific binding control. NE protein from nuclear extracts