Figure 8. ChIP assay demonstrates the direct binding of p-Smad3 on the promoter of miR-21 gene.

MCF-7 cells were pre-treated with 30 ng/ml TGFβ1 for 1 hour and immunoprecipitated by p-Smad3 antibody, RNA polymerase II antibody, or normal mouse IgG. The isolated DNA fragments were used for the templates in PCR analysis. SBS1 (ATGCATTCT) and SBS2 (AAGTCAGAGAG) are the reported binding sequences of p-Smad3. RNA polymerase II binding GAPDH promoter was used as a positive control system to confirm the efficiency of ChIP assay. The lengths of the PCR products including SBS1, SBS2, and GAPDH promoter sequences are 114bp, 100bp, and 166 bp, respectively.