FIGURE 2.

Perm1 encodes a protein found in multiple cellular compartments, is selectively expressed in muscle, and is regulated by physical activity. A, schematic representation of the human (h) and mouse (m) PERM1 proteins, showing the percent identity at the amino acid level and highlighting the conserved motifs (ØXXLL, protein interaction motif; NLS, nuclear localization signal; aa, amino acids) and the region recognized by the anti-PERM1 serum. B, detection of endogenous Perm1 in C2C12 myotubes infected with adenoviruses expressing shGFP (control), shPerm1, or PGC-1α on day 6 of differentiation. Cells were harvested on day 8, cell lysates were subjected to subcellular fractionation, and Perm1 protein was detected using the PERM1 antibody (α-PERM1). Endogenous Perm1 in control cells was detectable only after long exposure (second panel from top). Antibodies against cytoplasmic lactate dehydrogenase (LDH), mitochondrial HSP60, and nuclear lamins (three bottom panels) were used to assess the purity of the cytoplasmic (C), mitochondrial (M), and nuclear (N) fractions. Arrows indicate the two major protein isoforms encoded by Perm1; # indicates a nonspecific protein reacting with the antibody and enriched in the nuclear fraction. C, Perm1 mRNA levels in the indicated tissues of 10-week-old male mice (n = 3) were determined by RT-qPCR and normalized to levels of 36B4 in each tissue (Sol, soleus; EDL, extensor digitorum longus; GC, gastrocnemius). D, Perm1 protein in lysates from skeletal muscles, heart, brown adipose tissue (BAT), and liver of 10-week-old male mice was detected by SDS-PAGE and Western blot using the PERM1 antibody (upper panel); an antibody against GAPDH was used as a loading control (lower panel). E, Perm1 mRNA levels in the quadriceps muscles of 19-week-old female mice that had access to electronically monitored in-cage running wheels for 5 weeks (Trained; n = 7) and control sedentary littermate females (Control; n = 7) were determined by RT-qPCR, normalized to levels of GAPDH, and expressed relative to Perm1 levels in the control sedentary mice. F, PERM1 mRNA levels in muscle biopsies taken from male subjects (n = 7) before (Pre) or 3 h after (Post) an acute cycling bout (60 min at ∼70% of their VO₂ peak) were quantified by RT-qPCR as described (41). G, PERM1 mRNA levels in biopsies taken from male control subjects or ALS patients (n = 9) were quantified by RT-qPCR as described (24). Error bars represent S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.