Oxidative Medicine and Cellular Longevity / 2018 / Article / Fig 4

Research Article

p66Shc Inactivation Modifies RNS Production, Regulates Sirt3 Activity, and Improves Mitochondrial Homeostasis, Delaying the Aging Process in Mouse Brain

Figure 4

Effect of ONOO in Sirt3 activity. Recombinant Sirt3 (rSirt3) activity was examined using a fluorometric method. Assays were performed according to the manufacturer’s instructions. (a) Recombinant Sirt3 (rSirt3) was incubated with increased concentrations of ONOO (100, 250, and 500 μM). (b) Immunoprecipitation of Sirt3 was performed from mitochondrial brain lysates incubated with monoclonal anti-Sirt3 antibody and protein A/G PLUS Agarose and then were subjected to immunoblotting against monoclonal anti-Sirt3 or anti-3-nitrotyrosine antibodies. (c) rSirt3 was incubated with 100 μM ONOO or purified brain mitochondrial lysates from 3- and 24-month WT (grey) and p66Shc(−/−) (black) groups (, for each experimental group). Results were contrasted in pairs between rSirt3 and the two aged groups (a) as well as between both the 24-month-old WT and p66Shc(−/−) groups (b). Values represent ; A and B represent , one-way analysis of variance (ANOVA) and Bonferroni post hoc test.