p66Shc Inactivation Modifies RNS Production, Regulates Sirt3 Activity, and Improves Mitochondrial Homeostasis, Delaying the Aging Process in Mouse Brain
nNOS expression and RNS production in p66Shc(−/−) brain mice during aging. Mice were sacrificed at 3, 18, and 24 months of age. (a) Time course of nNOS activity in mitochondria brain mice measured by 3H-L-citrulline formation (). (b) RT-qPCR and immunoblot of proteins separated using SDS-PAGE from whole brain lysate reveals nNOS mRNA levels and protein expression of mouse brain in WT (grey) and p66Shc(−/−) (black) groups at 3 and 24 months of age. GADPH mRNA levels and actin protein expression were used as housekeeping control and loading control, respectively (, for each experimental group). (c) NO levels were obtained from 50 μg/mL of isolated mitochondria incubated with 0.3 mM L-arginine, 10 μM DAF-FM, and 0.5 μM MitoTracker (per duplicate) at 495 nm (excitation) and 515 nm (emission) in the presence or absence of the NOS inhibitor (3 mM L-NAME) from (). (d) Western blot of nitrated proteins from the different groups; membranes were revealed with anti-3-nitrotyrosine antibodies for each group (). Values represent error of the mean (SEM); A represents compared to the 3-month-old group, and B represents between 24-month-old WT and p66Shc(−/−) groups, one-way analysis of variance (ANOVA) and Bonferroni post hoc test.