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. Author manuscript; available in PMC: 2012 Jan 26.
Published in final edited form as: Mol Cell. 2010 Dec 22;40(6):893–904. doi: 10.1016/j.molcel.2010.12.013

Figure 3. Re-expression of Sirt3 in Sirt3−/− MEFs deacetylates MnSOD and restores SOD activity.

Figure 3

(a) Expression of wild-type, but not a deacetylation null mutant of Sirt3, deacetylates MnSOD. Third passage Sirt3−/− MEFs were transfected with a control lentivirus, virus expressing a wild-type Sirt3 (lenti-Sirt3-wt), or deacetylation null gene (lenti-Sirt3-dn). Forty hours after infection, Sirt3−/− MEFs were harvested and mitochondrial extracts were IPed with an anti-MnSOD antibody, separated into two equal fractions, and immunoblotted with either an anti-acetyl or anti-MnSOD antibody. (b) The mitochondrial samples above were also were also separated and immunoblotted with an anti-myc antibody (Santa Cruz Biotechnology, Inc). (c) Expression of wild-type, but not a deacetylation null mutant Sirt3 gene increases mitochondrial MnSOD activity. Whole cell homogenates from Sirt3−/−MEFs were infected with lenti-Sirt3-wt or lenti-Sirt3-dn and assayed for MnSOD activity in 50 mM potassium phosphate buffer. MnSOD activity was determined using an indirect competitive inhibition assay as described in the methods section (Spitz and Oberley, 1989). Results in this figure are the mean of at least three separate experiments and error bars represent one standard deviation. * indicates P < 0.05 by t-test. (d) Expression of wild-type, but not a deacetylation null mutant Sirt3 gene, decreases mitochondrial superoxide levels. Mitochondrial superoxide levels were determined, after infection as described above, by the addition of Mito-SOX (1 μM) to the cells followed by incubation for an additional 10 minutes before being trypsinized and resuspended. Fluorescence was measured via flow cytometry. (e) SIRT3 directly deacetylates MnSOD in vitro. IPed purified MnSOD was mixed without (lane 1) or with (lanes 2-4) recombinant human SIRT3 with NAD+ (lanes 2 and 4) or nicotinamide (lane 4) and incubated. Samples were separated followed by immunoblotting with an anti-acetyl antibody. Immunoblotting for SIRT3 and MnSOD were done as internal controls. (f) IPed purified MnSOD was mixed with recombinant human SIRT3 without and with NAD+ were separated and immunoblotted with an anti-K122-MnSOD antibody (Epitomics, Inc - The Rabbit Monoclonal Antibody Company.