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. 2016 Feb 20;24(6):312–328. doi: 10.1089/ars.2015.6403

FIG. 4.

FIG. 4.

Sirt3 deacetylates SOD2 at K130 and ATP synthase β at K485 to alleviate ROS accumulation and ATP depletion. (A, B) WT-SOD2, WT-ATP synthase β, and their mutants were cotransfected with Sirt3-EGFP into N-2a cells. The acetylation levels of SOD2, ATP synthase β, and their mutants were measured by Western blotting after immunoprecipitation with ANTI-FLAG M2 Affinity Gel. (C, D) WT-SOD2, WT-ATP synthase β, and their mutants were transfected into N-2a cells with 500 μM MPP+ for 24 h. The acetylation levels of SOD2, ATP synthase β, and their mutants were measured by Western blotting after immunoprecipitation with ANTI-FLAG M2 Affinity Gel. (E) ROS level was measured in N-2a cells transfected with WT-SOD2 and its mutants with 500 μM MPP+ for 24 h. Quantitative data = mean ± SEM, n = 3, **p < 0.01; ***p < 0.001, comparison against untreated, paired two-tailed Student's t-test. (F) ATP was measured in N-2a cells transfected with WT-ATP synthase β and its mutants with 500 μM MPP+ for 24 h. Quantitative data = mean ± SEM, n = 3, *p < 0.05, comparison against WT, ANOVA with Dunnett test. WT-SOD2, WT-ATP synthase β, and their mutants were transfected into N-2a cells with 5 mM NAM (G, H) or Sirt3 siRNA (I, J). Quantitative data = mean ± SEM, n = 3, **p < 0.01, comparison against control group, paired two-tailed Student's t test. The acetylation levels of SOD2, ATP synthase β, and their mutants were measured by Western blotting after immunoprecipitation with ANTI-FLAG M2 Affinity Gel. (K) Protein sequence alignment of SOD2 and ATP synthase β cross species was performed by UniProt. (L) The acetylation levels of SOD2 and ATP synthase β were measured by Western blotting with SOD-K130-Ac and synthase β-K485-Ac antibodies. N-2a cells were transfected with Sirt3-3×FLAG or Sirt3 siRNA, or treated with 500 μM MPP+. (M) The acetylation levels of SOD2 ATP synthase β in mouse primary midbrain neurons, rat primary midbrain neurons, and SH-SY5Y cells were measured by Western blotting under MPP+ treatment for 24 h. SH-SY5Y cells were treated with 500 μM MPP+, while the other two neurons were 50 μM MPP+. Quantitative data = mean ± SEM, n = 3, *p < 0.05; **p < 0.01, comparison against control group, paired two-tailed Student's t test. NAM, nicotinamide.