Figure 10.

Smad3 negatively regulates E2F3 transcription by binding to its promoter. (A) A map position of primers for ChIP PCR and ChIP RT-PCR. (B) Immunoprecipitation in mouse islets to show the specificity of Smad3 antibody used for ChIP assay. (C) ChIP PCR shows the binding of Smad3 on the promoter of E2F3 locus. The ChIP PCR product is analyzed by agarose electrophoresis. (D) RT-PCR is used to quantitate the ChIP efficiency of Smad3 antibody to precipitate E2F3 promoter DNA compared with the non-specific IgG isotype, which is normalized to input. (E) The structure of recombined firefly luciferase expression vector driven by the E2F3 promoter with normal or mutated Smad3 binding site. (F) Dual-luciferase reporter assay shows the transcriptional regulation of Smad3 on cloned E2F3 promoter in HEK293T cells. Each dot represents one independent experiment and data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 compared with control or as indicated.