Anti sod2 acetyl k68

Primary antibodies included anti-acetyl-lysine (Cell Signaling #9441), anti-SIRT3 (Cell Signaling #5490), anti-SOD2 (acetyl K68) (Abcam ab137037), anti-SOD2 (Cell Signaling #13141), anti-VDAC (Cell Signaling #4866), anti-GAPDH (Cell Signaling #5174), anti-FXN (generous gift of Grazia Isaya, Mayo Clinic, Rochester MN), anti-LCAD (Abcam ab196655) and to the electron transport chain complexes: anti-CI-NDUFA9 (Abcam ab14713), anti-CII-SDHB (Abcam ab14714) and anti-CIII-UQCRFS1 (Abcam ab14746). Band intensities were measured using ImageJ (IJ1.46). To determine correlation between acetylation and heart function, relative density of acetylation for n = 3 western blot images was normalized to day 30 controls and averages for each group were used for correlation calculation.

The brain tissues were lysed with mammalian protein extraction buffer (Pierce, Rockford, IL). Lysates of brain tissue or isolated mitochondria from four groups of mice were analyzed by western blot analysis as previously described^{60 (link)}. Briefly, samples with equal amount of protein (30–50 µg) were resolved on 7.5% or 12% tris glycine gels by SDS-PAGE. Separated proteins were transferred onto polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in 5% nonfat milk for 1 h at RT and then incubated with specific primary antibody (1:1000) at 4 °C for overnight. The antibodies against acetyl lysine (Cat # 9441), Sirt1 (Cat # 9475), Sirt3 (Cat # 5490), Sirt5 (Cat # 8782), Drp1 (Cat # 14647), Ac-p65 (#12629), p65 (#3034) and β actin (Cat # 4967) were from Cell Signaling Technology (CST, Danvers, MA). Anti-SOD2 (acetyl K68; Cat # ab137037), anti-Mfn1 (Cat # ab57602) and anti-Mfn2 (Cat # ab124773) and OXPHOS antibody cocktail (Cat # ab110412) were from AbCam (Cambridge, MA). After washing in TBST, the membranes were exposed to secondary antibodies conjugated to alkaline phosphatase and the signals were developed with CDP-Star reagent (Sigma Aldrich-St Louis, MO). The band intensities were measured using Fluor S MultiImager and Quantity One software from Bio-Rad and corrected for the levels of β actin.