Smad3 and Smad4 directly bind to and activate the MCP-1 promoter. (A) Smad3 and Smad4 enhanced the expression of the luciferase reporter under the control of the MCP-1 promoter region containing −793 to −521 bp. The reporter −793 to −521-MLP-Luc was cotransfected into HepG2 cells with various Smad proteins as indicated. The luciferase assay was performed similarly as in Figure 6. Error bars indicate SD. (B) Smad3 and Smad4 directly bound to the −793-bp to −521-bp fragment as shown by EMSA. Purified GST, GST-Smad3, or GST-Smad4 and ³²P-radiolabeled −793-bp to −521-bp fragment were incubated in the presence or absence of 20-fold excess unlabeled probe (cold probe). The DNA-protein complex was analyzed with a polyacrylamide gel and autoradiography. (C) There are 4 potential SBEs (bold and italic) in the −793-bp to −521-bp region. The SBE in probe A was mutated with C_-619 replaced by T to generate MT probe A. (D) Smad3 and Smad4 bound to probe A with a high affinity. EMSA was performed similarly as in panel B. (E) Mutation in the SBE box in probe A abolished Smad binding. (F) Smad binding was essential for the TGF-β–enhanced MCP-1 promoter activity. The reporters containing wild-type or mutant (as in the MT probe A) −793-bp to −521-bp fragment were transfected to HepG2 cells. After being treated with TGF-β1 for 16 hours later, the cells were harvested for luciferase measurement. Error bars indicate SD. (G) TGF-β enhanced the binding of endogenous Smad4 to the MCP-1 promoter. HMVECs were treated with TGF-β1 for 2 hours and subjected to ChIP with anti-Smad4 antibody. The Smad4-bound DNA was MCP-1 amplified by PCR with the primers for the promoter region −793 bp to −523 bp or for the 501 to 777 bp region downstream of ATG.