SIRT2 interacts with FOXO1 and deacetylates FOXO1. (A) HEK293 cells were transiently transfected with FOXO1, SIRT2-Flag, or both by the calcium phosphate method. Forty hours after transfection, cells were lysed, and SIRT2-Flag proteins were immunoprecipitated with agarose beads and conjugated with anti-Flag antibodies. The precipitated proteins were washed five times and analyzed by Western blotting as indicated. IP, immunoprecipitation. IB, immunoblotting. (B) HEK293 cells were transfected with Flag-FOXO1 and SIRT2. Cells were treated with 1 μM tricostatin A (TSA) for 2 h before harvest. Flag-FOXO1 was immunoprecipitated with anti-Flag beads, and acetylated FOXO1 was detected with anti-acetylated lysine antibodies. (C) Endogenous SIRT2 in 3T3-L1 adipocyte lysate was immunoprecipitated with anti-SIRT2 antibody, and coprecipitation of FOXO1 was detected by Western blot assay. (D) SIRT2 knockdown or vector control adipocytes were treated with 1 μM TSA for 2 h. Cells were lysed and endogenous FOXO1 was immunoprecipitated with anti-FOXO1 antibodies, and the acetylation level was detected by anti-acetyl lysine antibodies. AcK, acetylated lysine.