Figure 2. Acetylation negatively regulates PK, PEPCK protein levels and PGAM activity. (A) Acetylation analyses of PK, PGAM and PEPCK. GFP-PK-V5, GFP-PGAM-V5 or GFP-PEPCK-V5 plasmid was transfected into HzAm1 cells, followed by Sirt2 inhibitor NAM treatment. Protein acetylation was analyzed by immunoprecipitation (IP) and western blotting with anti-acetyl-lysine antibody. WCE: whole cell extracts. Changes in PK (B), PEPCK (C), and PGAM (D) protein levels in response to NAM treatment. (E) Changes in PGAM activity with NAM treatment. (F) Changes in pyruvate levels with NAM treatment. (G) Changes in TCA cycle activity with NAM treatment. HzAm1 cells were cultured in 0, 5, 10, and 15 mM NAM for 48 h. (H) Expression pattern of Sirt2 during pupal development. Protein extracts from the brains or cells were used for western blotting with the indicated antibodies. Protein bands were quantified using ImageJ software and normalized to the levels of H. armigera actin (5 μg). Pyruvate levels and enzyme activity was measured and normalized against total protein levels. DP, diapause-destined pupae; NP, nondiapause-destined pupae. (I) Developmental delay caused by AGK2 injection. Day-1 nondiapause-destined pupae were injected with 2 μg (n=60) or 4 μg (n=60) AGK2 or DMSO (n=60) as the control. Developmental delay was determined by examining the location of the pupal stemmata. Each point represents the means±S.D. of three independent replicates. *, p<0.05; **, p<0.01 (determined by independent t-test).