Fig. 3

Endurance exercise increases the abundance of p53 in mitochondrial matrix where it interacts with mtDNA in a complex with POLG1 and Tfam in mtDNA mutator mice. a Mitochondrial p53 is primarily localized in the matrix. Muscle mitochondria were subfractionated into outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), and matrix (Mx) fractions, and these fractions were immunoblotted for the compartment-specific proteins TOMM22 (~16 kDa), cytochrome c (~14 kDa), COX-IV (~17 kDa), and CS (~45 kDa), respectively, and also for p53 (~53 kDa) and Tfam (~24 kDa). Representative blots of b mitochondrial p53 content (~53 kDa) in the muscle and heart of WT, PolG-SED, PolG-END (n = 6–8/group), c p53 co-immunoprecipitation (IP) followed by immunoblotting (IB) for mitochondrial transcription factor A (Tfam; ~24 kDa) to assess mitochondrial p53-Tfam complex content in muscle and heart mitochondria from WT, PolG-SED, PolG-END (n = 4–5/group), and d p53 co-IP followed by IB for POLG1 (~140 kDa) to assess mitochondrial p53-POLG1 complex content in muscle and heart mitochondria from WT, PolG-SED, and PolG-END (n = 6–8/group). VDAC (~32 kDa) was used as a mitochondrial loading control. e p53-POLG1-Tfam complex is bound to mtDNA (quantified using two independent mtDNA regions: COX-II and cytochrome b) in muscle mitochondrial fractions of WT, PolG-SED, and PolG-END mice (n = 4–6/group). A non-specific IgG antibody was used as negative control antibody. Asterisk (PolG-SED vs. both WT and PolG-END): *P < 0.05, **P < 0.01; dagger (PolG-END vs. WT): ^† P < 0.05. Error bars represent SEM. AU arbitrary units