Figure 3

Endothelin-1 (ET-1) decreased the binding of Fli1 to the human α2 (I) collagen (COL1A2) promoter via increasing its phosphorylation at threonine 312. ( A ) Normal dermal fibroblasts were treated with ET-1 for the indicated period of time. Whole cell lysates were subjected to immunoblotting with anti-p-Fli1 antibody. To determine the total Fli1 levels, nuclear extracts were used for immunoblotting with anti-Fli1 antibody. ( B ) Nuclear extracts were incubated with biotin-labeled oligonucleotides. Proteins bound to these nucleotides were isolated with streptavidin-agarose beads, and Fli1 was detected by immunoblotting. Total Fli1 protein levels were determined by immunoblotting using the same nuclear extracts. ( C ) Chromatin was isolated from normal dermal fibroblasts and immunoprecipitated using rabbit anti-Fli1 antibody or rabbit IgG. After isolation of bound DNA, PCR amplification was carried out using COL1A2 promoter-specific primers. Input DNA was taken from each sample before addition of an antibody. The cycle threshold (Ct) values from IgG and Fli1 pull-down were subtracted by the number obtained from 10 × diluted input DNA. The relative pull-down of Fli1 was then normalized by the subtracted Ct value of IgG. The mean value of normal dermal fibroblasts without ET-1 stimulation was arbitrarily set at 1. All bands show one representative of three independent experiments. DNAP, DNA affinity precipitation; AU, arbitrary unit.