Fig. 1

Sirt1 interacts with resting and active Smad2 in vitro and in vivo. a Hep3B cells transfected with HA-Sirt1 and either Flag-Smad2 or EF-Flag were treated with TSA for 8 h followed by TGFβ for the indicated times. Smad2 was purified by immunoprecipitation and Sirt1 or Smad2 were detected by Western Blot. Fold increase (lane 4–6 over lane 4) of HA/Flag levels are indicated at the bottom. Weak Sirt1 binding to control samples were taken into account for calculations. b HEK293T cells transfected with Flag-Smad2 were treated overnight with SB-431542, then washed out and treated with TGFβ for 1 h. Flag-Smad2 was purified with a flag peptide competitor and used for His-Pull-down assay. Flag-Smad2, PSmad2 and Sirt1-6xHis were detected by Western Blot. Fold increase (lane 2 and 4 over lane 2) of Flag/HIS levels are indicated at the bottom. The vertical lines across the blot indicate that two distant parts of the very same blot were put together. Lanes are considered left to right. All the experiments of this figure were repeated at least three times. Representative results are shown. IP: Immunoprecipitation