Fig. 5.

ARNT mediates transcriptional effect of c-Jun on promoter activation of 12(S)-lipoxygenase gene. a Cells were transfected with pXLO-7-1, myc-ARNT and TAM-67 by lipofection. The luciferase activities and protein concentrations were then determined and normalized. Values represent means ± SEM of three determinations. Expression of myc-ARNT and TAM-67 proteins were analyzed by western blot analysis using anti-ARNT and anti-c-Jun antibodies. b Cells were transfected with TAM-67 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 TAM-67 to Sp1 probes was analyzed by western blot. c Cells were transfected with 20 nM ARNT siRNA and scramble (SC) oligonucleotides and then treated with 50 ng/ml EGF for 3 h. Nuclear extracts were prepared and the Sp1, ARNT and c-Jun proteins were detected by anti-Sp1, anti-ARNT, anti-phospho-c-Jun (Ser 73) and anti-c-Jun antibodies, respectively. d Nuclear extracts were immunoprecipitated (IP) with antibodies (Ab) against Sp1. The proteins were subjected to SDS–PAGE and analyzed by western blotting with antibodies against c-Jun, ARNT and Sp1. IgG indicates the negative control of antibodies