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. 2005 Oct;16(10):4623–4635. doi: 10.1091/mbc.E05-01-0033

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Copyright © 2005, The American Society for Cell Biology

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Figure 3. — Mitochondrial localization of SIRT3, SIRT4, and SIRT5. (A and B) The V5-His-tagged SIRT3, SIRT4, or SIRT5 was transiently expressed in NHF (A) and HeLa (B). The tagged proteins were detected by immunostaining with anti-V5 antibody. MitoTracker staining and DAPI staining are shown in parallel. (C) Cytoplasmic (Cyt), nuclear (Nuc), and mitochondrial (Mito) proteins were fractionated from 293T cells expressing V5-His-tagged SIRT3 (left), SIRT4 (middle), or SIRT5 (right), and analyzed in Western blot using anti-V5 antibody. Cytochrome C, a well known mitochondrial protein, was detected as a control. The gels stained with SimplyBlue SafeStain (Invitrogen) are also shown. Residual DNA in nuclear fractions gave the stronger staining. The presence of two SIRT3 bands is due to its N-terminal processing (Schwer et al., 2002). The exact nature of multiple SIRT4 bands is not known.