FIGURE 3.

Assay of the presence of SIRT1 and PGC-1α in mitochondrial native nucleoids. Native nucleoids were obtained from isolated liver mitochondria as described under “Experimental Procedures.” After sucrose gradient, nucleoid-containing fractions were identified by detecting the presence of TFAM by dot blot (using goat anti-TFAM) and mtDNA by PCR analysis of the D-loop region. Western blot analyses with mouse anti-cytochrome c oxidase subunit IV (COX IV), mouse anti-cytochrome c (Cyt c), rabbit anti-SOD2, and rabbit anti-HSP60 were carried out to determine the possible presence of protein contaminants. Fractions 6–9 were considered to contain native nucleoids due to the absence of protein contaminants and to the presence of TFAM and mtDNA. PGC-1α and SIRT1 were detected by using Santa Cruz Biotechnology antibodies. Immunoblots reported are from one experiment representative of at least three that gave similar results.