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. 2008 Aug 18;28(20):6384–6401. doi: 10.1128/MCB.00426-08

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FIG. 5. — SIRT3 is localized in the cytoplasm and mitochondria as well as nuclei of heart tissue. (A) Nuclear (Nucl.) and nonnuclear (Non Nucl.) fractions of mouse heart were prepared by using the Pierce cell fractionation kit. Fractions were characterized by Western blotting taking GAPDH and MnSOD as nonnuclear markers and RNA polymerase II as a nuclear marker. Both fractions were analyzed for the expression level of SIRT3 by Western blotting. Actin was utilized as a loading control. Note the presence of the (∼44-kDa) long form of SIRT3 in both the nuclear and nonnuclear fractions, while the ∼28-kDa short form is detected only in the nonnuclear fraction. (B) Three subcellular fractions (cytoplasmic, mitochondrial, and nuclear) of a mouse heart were prepared, utilizing the Calbiochem fractionation kit, and characterized by Western blotting using fraction-specific protein antibodies. All three fractions were also analyzed for expression of mSIRT3 by Western blotting. The SIRT3 antibody Ab56214 was used in these blotting assays. Note the presence of ∼44-kDa SIRT3 in all three fractions, whereas the short form (∼28 kDa) of SIRT3 was detected only in the mitochondrial fraction of the heart.