FIGURE 4.

Ski interacts with SIRT1 and enhances SIRT1-promoted p53 deacetylation. A, Ski-mediated p53 deacetylation is blocked by treatment with nicotinamide. H1299 cells were transfected with the indicated constructs and treated with 10 mm nicotinamide for 12 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. B, SIRT1 knockdown abrogates Ski-mediated p53 deacetylation. H1299 cells were transfected with the indicated constructs and siRNAs. The cell lysates were analyzed by immunoblotting with the indicated antibodies. C, endogenous Ski and SIRT1 interact in MCF7 cells. The cell lysates were immunoprecipitated (IP) with normal goat IgG or an anti-Ski antibody and then blotted for SIRT1 or Ski. D, Ski promotes p53-SIRT1 binding. H1299 cells were transfected with the indicated constructs, and FLAG-p53 was immunoprecipitated with anti-FLAG antibodies. The co-precipitated SIRT1 was detected by immunoblotting with an anti-SIRT1 antibody. E, Ski and SIRT1 cooperate to inhibit p53 acetylation. H1299 cells were transfected with the indicated constructs, and the levels of acetylated p53 were determined by immunoblotting with an anti-acetylated p53 antibody. F, Ski knockdown abrogates SIRT1-mediated p53 deacetylation. H1299 cells were transfected with the indicated constructs and siRNAs. The cell lysates were analyzed by immunoblotting with the indicated antibodies. G, SIRT1 knockdown abolishes Ski-mediated repression of p53 transcriptional activity. H1299 cells were transfected with pp53-TA-Luc in combination with the indicated constructs and siRNAs. After 24 h, luciferase activity was measured. The experiment was performed in triplicate, and the data are represented as mean -fold activation ± S.D.