Figure 4. PARK2-mediated mitophagy is required for the activation of 26S proteasomes in denervated soleus muscle. (A) Western blot analysis of soleus muscles. Whole-tissue lysates of soleus muscles were immunoblotted with antibodies against the indicated proteins. The data shown are representative of at least 3 separate experiments. (B) Quantification of the mRNA levels for atrophy-related E3 ubiquitin ligases (Fbxo32 and Trim63) in soleus muscles by real-time PCR. Data are shown as the ratios (mean ± s.d.) to the mRNA levels obtained from innervated soleus muscles from control mice. **P < 0.01. (C) Peptide hydrolysis activity of 26S proteasomes. Soleus muscle homogenates from Atg7 KO, Park2 KO, and control mice were used to assay the chymotryptic activity of proteasomes using Suc-LLVY-AMC as a substrate in the absence or presence of 20 µM epoxomicin. Data are shown as the percentage of the activity (mean ± s.d.) obtained from innervated soleus muscles from control mice. **P < 0.01, NS; not significant. (D) Quantitative densitometry of immunoblotting data for the proteasome subunits shown in a. *P < 0.05, **P < 0.01. (E) Quantification of the mRNA levels of proteasome subunits in soleus muscles by real-time PCR. Data are shown as the ratios (mean ± s.d.) to the mRNA levels obtained from innervated soleus muscles from control mice. *P < 0.05, **P < 0.01 vs denervated muscle from control mice. (F) Nuclear levels of NFE2L1 in soleus muscles. Nuclear extracts prepared from denervated and innervated soleus muscles and total tissue lysates were immunoblotted with anti-NFE2L1, anti- NFE2L2, anti-LMNB (as a loading control for nuclear extracts), and GAPDH (as a loading control for tissue lysates) antibodies. The data shown are representative of at least 3 separate experiments.