Molecular determinants of the profibrogenic effects of endothelin-1 in pancreatic stellate cells

Figure 6 Effects of TGF-β1 and TNF-α on protein binding to the ET-1 promoter. Serum-starved PSC growing in 6-well plates were incubated with (A) TGF-β1 (5 μg/L) and (B) TNF-α (10 μg/L) for the indicated periods of time. Nuclear extracts were subjected to EMSA analysis using [³²P]-labelled oligonucleotides derived from the ET-1 promoter as described in the “Materials and Methods” section. Supershift analysis was performed by incubating binding reactions with the indicated protein-specific antibodies. Inducible complexes and supershifted bands (lane 6 in A and B) are pointed out by arrows. Addition of c-Rel antibody (B, lane 7) weakened protein binding to the oligonucleotide, suggesting the presence of the c-Rel protein in the complex (competitive binding of antibody and oligonucleotide to c-Rel). Results in (A) and (B) are representative of three independent experiments.