Research Paper Volume 10, Issue 5 pp 1053—1072

Deacetylation of metabolic enzymes by Sirt2 modulates pyruvate homeostasis to extend insect lifespan

Figure 4. Sirt2 interacts with and increases PGAM activity. (A) Sirt2 physically associates with PGAM. HzAm1 cells were co-transfected with GFP-Sirt2 and GFP-PGAM-V5 plasmids for 48 h, and then the cell extracts were immunoprecipitated (IP) with anti-V5 or anti-Sirt2 antibody, followed by immunoblotting (IB) with an anti-Sirt2 or anti-V5 antibody, respectively. WCE: whole cell extracts. (B) Sirt2 overexpression increases PGAM activity 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 GFP-Sirt2 plasmid. (C) Sirt2 knockdown decreases PGAM activity in vitro. (a) Dose-dependent response to Sirt2 RNAi. HzAm1 cells were transfected with Sirt2 dsRNA 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 PGAM activity. (a) Dose-dependent response to AGK2 treatment. HzAm1 cells were cultured with AGK2 for 48 h. (b) Time-dependent response to AGK2 treatment. HzAm1 cells were cultured with 10 μM AGK2. Enzyme activity was measured and normalized against total protein levels. Each point represents the means±S.D. of three independent replicates. *, p<0.05; **, p<0.01 (determined by independent t-test).