Figure 3.

SIRT2 deacetylates Foxo3a, increases Bim levels and leads to apoptosis in MPTP-treated mouse brains. (A) SIRT2 deacetylates Foxo3a in MPTP-injected mouse brains. The lysates from wt or SIRT2 KO mouse brains were immunoprecipitated with Foxo3a antibody or NRS (Normal Rabbit Serum) and blotted with anti-Foxo3a or anti-acetylated lysine (Ac-K) antibodies in saline or MPTP-injected mouse brains. Quantification of acetylated bands was carried out by densitometry using the NIH ImageJ program and is shown below the gel. Representative blots are shown. (B) Bim RNA levels quantified from whole brains of mice by qPCR. n = 6 for each indicated group. Statistical analyses were carried out using Two-Way ANOVA. ^*p < 0.01, wt-MPTP vs. wt-saline. (C) Western blotting of Bim protein extracted from whole brains of saline or MPTP-treated wt or SIRT2 KO mouse brains. n = 6 for each group. Actin serves as loading control. Representative immunoblot is shown. Quantification of the relative Bim protein levels is shown on the right. Statistical analyses were carried out using Two-Way ANOVA. ^*p < 0.01, wt-MPTP vs. wt-saline. (D) Western blotting of Bim levels from the lysates of SH-SY5Y cells that are transfected with Bim or Bim-shRNA plasmids. Actin serves as a loading control. (E) Caspase-3 activity of SH-SY5Y cells transfected with SIRT2, SIRT2-shRNA, Bim or Bim-shRNA plasmids and treated with 16 h MPP⁺. The graph on the right shows Caspase-3 activity of SH-SY5Y cells after Bim overexpression without MPP⁺ treatment. (F) Left panel shows the western blotting of SIRT2 protein extracted from wt or MPP⁺-treated SH-SY5Y cells. Three independent experiments were performed. Right panel shows the western blotting of SIRT2 protein extracted from whole brains of saline or MPTP-treated wt mice. n = 6 for each group. Actin serves as loading control. Representative immunoblots are shown. Quantifications of the relative SIRT2 protein levels are shown below. Statistical analyses were carried out using Two-Way ANOVA.