Research Paper Volume 6, Issue 10 pp 820—834

Figure 2. SIRT-1 dysregulation in aged mice increases skeletal muscle fatigue. Total RNA and cell lysates were isolated from the control or exercised gastrocnemius muscles. (A) Global cellular protein PARylation was determined in total cell lysate by immunoblots. (B) NAD+ levels in skeletal muscle were determined in control and exercised muscles. PARP-1 (C) and SIRT-1 (D) mRNA (top) and protein (bottom) levels were determined in total muscle mRNA and cell lysates, respectively. GAPDH was used as a loading control. (E) PGC-1α acetylation levels were estimated by immunoblotting after IP. (F) SIRT-1 and PARP-1 binding assays, PARP-1 acetylation levels (G) or PARP-1 and GCN5 binding assays (H) were estimated by immunoblotting after IP. (I) GCN5 mRNA (top) and protein (bottom) levels were determined in total muscle mRNA and cell lysate, respectively. (J) mRNA expression of the indicated genes in the total RNA was examined by qPCR. (K) Maximal evoked isometric forces from 350 contractions are shown for the plantar flexor muscles in young and aged mice. All force measurements were normalized to body weight (g). The blots are representative of three independent experiments. The data are presented as mean ± SEM (n = 3). White and black bars indicate non-exercised and exercised gastrocnemius muscles, respectively. COX17, cyclooxygenase 17; CytC, Cytochrome C; ERR-α, estrogen-related receptor α; mtTFA, mitochondrial transcription factor A; Ndufa2, NADH dehydrogenase [ubiquinone] iron-sulfur protein a 2; NRF1,nuclear respiratory factor 1; MCD, medium-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; SDH, succinate dehydrogenase; Tropn I, troponin I; UCP2, uncoupling protein 2; immunoprecipitation (IP).