Figure 6

SIRT1 deacetylates zyxin in vitro and in vivo. (A) SIRT1 deacetylates zyxin in vitro. GFP-zyxin, immunoprecipitated using anti-GFP antibody, was added with recombinant GST-SIRT1 in the presence or absence of NAD or nicotinamide (NAm). The acetylation levels of zyxin were determined using anti-acetylated lysine antibody. (B) SIRT1 deacetylates zyxin in mammalian cells. COS-7 cells were transfected with plasmids expressing GFP-zyxin and Myc-SIRT1 and incubated for 6 h in the presence or absence of leptomycin B. GFP-Zyxin was immunoprecipitated with anti-GFP antibody, and the acetylation levels of zyxin were determined using anti-acetylated lysine antibody. (C) SIRT1 H363Y does not affect the acetylation levels of zyxin. COS-7 cells were transfected with plasmids expressing GFP-zyxin and Myc-SIRT1 H363Y. GFP-Zyxin was immunoprecipitated with anti-GFP antibody, and the acetylation levels of zyxin were determined using anti-acetylated lysine antibody. (D) HEK 293T cells expressing GFP-zyxin were treated for 24 h in the presence or absence of NAm. GFP-zyxin was immunoprecipitated using GFP antibody, and the acetylation levels of zyxin were determined using anti-acetylated lysine antibody.