Figure 5

SIRT1 interacts with Smad4. (A) Smad4 binds to SIRT1 in OSCC cells. Nuclear extracts from OECM1 and HSC3 cells were immunoprecipitated using a SIRT1 or Smad4 antibody, and analyzed by western blot using antibodies against Smad2, Smad4, and SIRT1. (B) Knock down of SIRT1 increased endogenous Smad4 acetylation. Acetylated endogenous Smad4 proteins in OSCC cells with or without siSIRT1 were then immunopurified with Smad4 antibody. Western blots were probed with SIRT1, acetylated-Lysine (Ac-K), and Smad4. (C) Ectopic expression of SIRT1 reduced levels of acetylated Smad4. Equal amounts of protein (20 ug) from OECM1 cells were transient transfected with pEGFP-SIRT1 or vector alone (pEGFP-C1) for 0–48 h and analyzed by Western blot. (D) Western blotting revealed the expression and acetylation levels of endogenous Smad4 in OECM1 cell lines transient transfected with pEGFP-SIRT1 or vector alone (pEGFP-C1) for 24 h, and treated with TGF-β 5 ng/mL for 48 h. Western blots were probed with SIRT1, acetylated-Lysine (Ac-K), and Smad4.