FIGURE 9.

Acetylation of Smad3 participates in TGF-β1-induced Zp activation. A, schematic representation of the Smad3 protein and its mutant used in transient transfection experiments. B, the DG75 cells were transiently transfected with reporter gene construct containing the wild type Zp (−234 to +12) inserted upstream of the CAT gene (Control), or with a combination of expression plasmids for wild type of Smad3 (Smad3 wt) or its mutant (Smad3 mut), CBP, or empty vectors (pcDNA3.1 and pCMV2N3T) as indicated. The positive control consisted of Zp (−234 to +12) transfected DG75 cells and treated by 20 ng/ml of phorbol 12-myristate 13-acetate (PMA) immediately following transfection. Twenty-four hours later, the cells were harvested and lysed, and CAT activity was quantified as described under “Experimental Procedures.” The error bars represent the standard deviation. Significant differences between control and the other samples were determined by Student's t test; *, p < 0.05. C, cell lysates were immunoprecipitated (IP) with anti-Smad3 antibody, and equal amounts of immunoprecipitated Smad3 were analyzed by Western blotting with anti-acetyllysine antibodies.