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