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. 2007 Apr 16;27(12):4488–4499. doi: 10.1128/MCB.01636-06

FIG. 7.

FIG. 7.

Smad7 binds to DNA via its MH2 domain. (A) Smad7 binds to the ARE oligonucleotide via its MH2 domain. HEK293T cells were transfected with expression plasmids as indicated and treated with 100 pM TGF-β1 for 2 h. The cell lysates were harvested for oligonucleotide precipitation assay with the biotin-labeled 51-bp ARE oligonucleotide. DNA-bound proteins (top) and total protein levels (middle and bottom) were analyzed by anti-Flag or anti-Myc immunoblotting. FL, Full length. (B) Smad7 binds to ARE as confirmed by EMSA. HEK293T cells were transfected with Flag-Smad7 and treated with 100 pM TGF-β1 for 2 h. Nuclear extracts were used to perform gel mobility shift assays by using the ARE oligonucleotide as a probe in the presence of anti-Smad7 antibody or preimmune antiserum as indicated. The asterisk above lane 5 indicates that the probe was incubated with anti-Smad7 antibody in the absence of any nuclear extract. (C) Sequence of the ARE of the Mix.2 promoter. The SBE and FoxH1 binding sites are underlined. The dots indicate the mutated nucleotides in SBE. (D) The Smad7 MH2 domain specifically binds to the ARE, but not to its mutant (mut) sequence. GST fused to N-terminal Smad7 MH2 was expressed and purified from Escherichia coli. The oligonucleotide precipitation assay was carried out as described for panel A. (E) The binding of GST-Smad7 protein to DNA is specific. GST-Smad7 protein was purified from E. coli. EMSA was carried out using ARE, PAI-1 sequence, and their mutants. (F) Smad7 associates with the PAI-1 promoter in Hep3B cells under physiological conditions. A ChIP assay was performed with anti-Smad7 antibody. PCR amplification of the distal PAI-1 promoter (−744/−522) was performed to detect Smad7-bound DNA (anti-Smad7 ChIP). DNA input and Smad7 expression are shown. β-Actin acted as a negative control. TGF-β1-induced PAI-1 mRNA expression was detected by RT-PCR in Hep3B cells (right). Total RNA was isolated from Hep3B cells treated with TGF-β1 (100 pM) for 3 h. GAPDH mRNA expression served as an RT-PCR control. IgG, immunoglobulin G.