Research Paper Volume 6, Issue 10 pp 820—834

Figure 3. PARP-1 inhibition increases SIRT-1 activity and improves skeletal muscle fatigue. Total RNA and cell lysates were isolated from the skeletal muscle of mice that were exercised and PBS injected (Ex) or exercised and PJ34 injected (Ex + PJ34). (A) Global cellular protein PARylation was determined in total cell lysates by immunoblots. (B) NAD⁺ levels were determined in in skeletal muscle of Ex or Ex + PJ34 mice. (C) PGC-1α acetylation levels were estimated by immunoblotting after IP. PARP-1 (D) and SIRT-1 (E) mRNA (top) and protein (bottom) levels were determined in total muscle mRNA and cell lysates, respectively. GAPDH was used as a loading control. PARP-1 acetylation levels (F), SIRT-1 and PARP-1 binding assays (G), or PARP-1 and GCN5 binding assays (H) were estimated by immunoblotting after IP. (I) mRNA expression of the indicated genes in the total RNA was examined by qPCR. (J) mtDNA was evaluated in total muscle genomic DNA by qPCR. (K) Maximal isometric forces from the plantar flexor muscles are presented for Ex or Ex + PJ34 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 Ex and Ex + PJ34 mice, 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. IP, immunoprecipitation.