Fig. 6.
ARNT is essential for c-Jun/Sp1-regulated gene expression. c4 cells (a) and vT2 cells (b) were transfected with pXLO-7-1 of 12(S)-lipoxygenase gene promoter, pPLA599 of cPLA 2 gene promoter and pRSVjun by lipofection. The luciferase activities and protein concentrations were then determined and normalized. Values represent means ± SEM of three determinations. c A431 cells were transfected with 50 nM ARNT siRNA or scramble(SC ) oligonucleotides by lipofection. After EGF treatment for 4 h, total RNA was extracted for reverse transcription PCR with p21WAF1/CIP1, Skp2, ARNT and GAPDH primers. d A431 cells were transfected with 20 or 100 nM ARNT siRNA or scramble (SC) oligonucleotides by lipofection. After EGF treatment for 4 h, the p21WAF1/CIP1, ARNT and Skp2 proteins were detected by anti-p21WAF1/CIP1, anti-ARNT and anti-Skp2 antibodies, respectively. e Cells were starved for 18 h in serum-free culture medium and then treated with 50 ng/ml EGF for 3 h. Nuclear extracts were prepared and DNA affinity precipitation assay was performed. Binding of Sp1, ARNT and c-Jun to Sp1 binding site of p21WAF1/CIP1 promoter (p21-Sp1) was analyzed by western blot. The streptavidin-agarose beads were used as a nonspecific binding control. f Cells were transfected with 20 nM ARNT siRNA by lipofection. After EGF treatment for 3 h, nuclear extracts were prepared, chromatin immunoprecipitation (ChIP, right panel), and DNA affinity precipitation assay (DAPA, left panel) was performed as described under “Materials and methods.” Binding of Sp1 and ARNT to Sp1 site of p21WAF1/CIP1 promoter region (p21-Sp1) was analyzed by western blot. The streptavidin-agarose beads were used as a nonspecific binding control. NE protein from nuclear extracts