Figure 5. Sirt2 interacts with and stabilizes PEPCK levels. (A) Sirt2 directly binds to PEPCK. HzAm1 cells were co-transfected with GFP-Sirt2 and GFP-PEPCK-V5 plasmids for 48 h, and the cell extracts were immunoprecipitated (IP) with anti-V5 or anti-Sirt2 antibody, followed by immunoblotting (IB) with anti-Sirt2 or anti-V5 antibody, respectively. WCE: whole cell extracts. (B) Sirt2 transfection increases endogenous PEPCK protein levels in vitro. (a) Dose-dependent response to Sirt2 overexpression. HzAm1 cells were transfected with GFP-Sirt2 or GFP-V5 plasmid for 48 h. (b) Time-dependent response to Sirt2 transfection. HzAm1 cells were transfected with 1.5 μg of recombinant Sirt2 plasmid. (C) Sirt2 knockdown decreases endogenous PEPCK protein levels in vitro. (a) Dose-dependent response to Sirt2 RNAi. HzAm cells were transfected with Sirt2 or GFP dsRNA for 48 h. (b) Time-dependent response to RNAi. HzAm1 cells were transfected with 4 μg Sirt2 dsRNA. (D) Effects of Sirt2 inhibitor AGK2 on PEPCK protein levels. (a) Dose-dependent response to AGK2 treatment. HzAm1 cells were cultured with AGK2 for 48 h. (b) Time-dependent response to AGK2 treatment. HzAm cells were cultured with 10 μM AGK2. Proteins were extracted from the cells for IB with anti-PEPCK antibody. Protein bands were quantified using ImageJ software and normalized to the levels of H. armigera actin (5 μg). Each point represents the means±S.D. of three independent replicates. *, p<0.05; **, p<0.01 (determined by independent t-test).