Fig. 2.

ARNT transactivates the promoter activity of 12(S)-lipoxygenase. a Cells were transfected with wild-type 12(S)-lipoxygenase promoter (pXLO-7-1), and myc-ARNT expression vector by lipofection. Cells were treated with 50 ng/ml EGF and further cultured in fresh medium up to 18 h. The luciferase activities and protein concentrations were then determined and normalized. Values represent means ± SEM of three determinations. b Cells were transfected with ARNT expression vector, wild-type (pXLO-7-1) and Sp1 sites mutant (SPM7) of 12(S)-lipoxygenase promoter by lipofection. The luciferase activities and protein concentrations were then determined and normalized. Values represent means ± SEM of three determinations. c Cells were transfected with 20 nM Sp1 siRNA by lipofection. After EGF treatment for 3 h, nuclear extracts were prepared, DNA affinity precipitation assay (DAPA, right panel) was performed. Binding of Sp1 and ARNT 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