Fig. 4. SIRT1 interacts with p53 in vitro and in vivo. (A) Pull-down experiment performed with in vitro translated (IVT) SIRT1 and GST–p53 (lane 3) or different GST–p53 fusions expressing different portions of the protein as indicated (lanes 4–6). Complexes were resolved by SDS–PAGE and visualized by autoradiography. IVT SIRT1 is indicated by an arrow. The exposure time of the input lane is half that of the binding reactions. (B and C) Whole-cell extracts (WCE) from untreated 293T cells or HCT116 UV-irradiated cells (20J/m2 for 5 or 20 h) were immunoprecipitated (IP) with either anti-SIRT1 or pre-immune sera, and the immunocomplexes analyzed by western blotting with anti-p53 (DO-1) antibody. Different exposures are shown in (C) in order to visualize p53 immunoprecipitated in the various samples. The asterisks indicate the Ig heavy chain.