TGF-β1 induces nuclear protein binding to the Ant1 TGF-β1 RE. A. Sequence of the Ant1 promoter containing the TGF-β1 response element (Ant1 Prom3-5'). Boxed sequence indicates the CAAT and TATA boxes with the identified consensus Sp1 binding sites and putative SBE shown in red. Sequences that were tested in the current studies are underlined. Oligonucleotides used in EMSAs are noted in bold-face type with the corresponding promoter sequence. B. Nuclear extracts were prepared from untreated (0) primary astrocyte cultures and those treated with TGF-β1 for one (1) and three (3) hours. Extracts were then incubated with Oligo 1. TGF-β1 induced nuclear protein binding to this oligonucleotide (arrows on left side). Nuclear extracts from untransfected primary cultured astrocytes treated with TGF-β1 for three hours were incubated with Oligo 1 and an antibody against Sp1 (S). The arrow on the right side indicates the supershifted species. C. Similar EMSAs were conducted using the same nuclear extracts with Oligo 2 (left panel) and Oligo 1Δ (right panel). Although specific nuclear protein binding is detected for these oligonucletide probes, a TGF-β1 induced species was not identified in either case. Control EMSA reactions were either conducted in the presence of unlabelled oligonucleotide competitors (C) or in the absence of nuclear extracts (F).