Lipid Peroxidation Red and white gastrocnemius was weighed, then minced and homogenized using a glass-Teflon Porter–Elvenhejm homogenizer (4% wt/vol) in buffer containing 50 mM dibasic potassium phosphate, 5 mM EDTA, 0.5 mM DTT, 1.15% KCl and 1 mM butylated hydroxytoluene at pH 7.4. The homogenates were centrifuged at 600 g for 10 min at 4uC. The resulting supernatant was decanted and protein concentrations of the spun muscle homogenates were determined using the Lowry Protein Assay. The homogenates were then assayed for malondialdehyde (MDA) according to Esterbauer and Cheeseman [14]. The absorbance of the supernatant was measured at 586 nm on an ultraviolet spectrophotometer (Cecil 9200 Super Aquarius, Cam- bridge, UK). A stock solution containing 10 mM 1,1,3,3,- tetramethoxypropane was used to obtain a standard curve of the following concentrations 0, 0.5, 1.0, 1.5, 2.5 and 5.0 mM, and the MDA concentrations in our samples were determined using the standard curve. All samples were analyzed in duplicate. MDA concentrations were expressed as nmol/g wet weight tissue. Protein Oxidation Carbonyl groups were measured in red and white gastrocnemius using the Zentech Protein Carbonyl (PC) enzyme immunoassay kit (Zenith Technology, Dunedin, New Zealand). Samples containing protein were reacted with DNP, and nonspecifically adsorbed to an ELISA plate. Unconjugated DNP and nonprotein constituents were washed and the adsorbed protein was probed with a commercial biotinylated anti-DNP antibody followed by strepta- vidin-linked horseradish peroxidase. Absorbances were related to a standard curve (0, 0.1, 0.23, 0.46 and 1.07 nmol/mg) prepared of BSA containing increasing proportions of hypochlorous acid (HOCl)-oxidized protein that was calibrated colorimetrically. Absorbance was read at 450 nm on a fluorescence plate reader. Protein carbonyl concentrations were expressed as nmol/mg. Catalase Activity Muscle catalase activity was determined by measuring the kinetic decomposition of H2O2, according to Aebi et al (1984) [15]. White gastrocnemius (50 ml) was added to an optical glass cuvette containing 930 ml of phosphate buffer (50 mM phosphate with 5 mM EDTA, and 0.1% Triton X-100 at pH 7.4). Then, 20 ml of 1 M H2O2 were added to the cuvette and mixed with a Pasteur pipette to initiate the reaction. Absorbance was measured at 240 nm for 2 min at 25uC. Catalase activity was calculated using the molar extinction coefficient for H2O2 of 0.0394 mmol/ ml, and expressed in mmol H2O2/min/wet weight tissue. Citrate Synthase and Cytochrome c Oxidase Activity Muscle lysate citrate synthase (EC 2.3.3.1) activity was deter- mined by measuring the formation of thionitrobenzoate anion, as previously described [16]. Absorbance was recorded at 412 nm every 30 s for 3 min at 37uC. CS activity is expressed in nmol/min/ mg of protein. Mitochondrial electron transport chain complex c oxidase (EC 1.9.3.1) activity was determined by measuring the rate of oxidation of reduced cytochrome c oxidase, as previously described [16]. Absorbance was recorded at 550 nm every 30 s for 3 min at 37uC. All samples were analyzed in duplicate on a spectrophotometer (Cary Bio-300, Varion, Inc., Palo Alto, CA). Western Blotting Equal amounts of protein were size-separated by an 8–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and were transferred to nitrocellulose membranes (#165- Caloric Restriction and ALS 3322, Bio-Rad Mini-PROTEAN 3 electrophoresis system, Mis- sissauga, ON, Canada) at 100 V. The membranes were blocked with 5% fat free milk or 3% BSA diluted in Tris-buffered saline with tween (0.5%) for 1 h at room temperature and incubated with primary antibodies against cytochrome c oxidase subunit I (COX- I; dilution 1:1000; MS404, Mitosciences, Eugene, OR), cyto- chrome c oxidase subunit IV (COX-IV, 1:3000; MS407, Mitosciences), citrate synthase (CS, 1:4000; a generous gift from Dr. Brian Robinson, The Hospital for Sick Children, Toronto, ON), manganese superoxide dismutate (MnSOD, 1:8000; ab13534, Abcam Inc., Cambridge, MA), copper/zinc superoxide dismuate (Cu/Zn-SOD, 1:8000; ab16831, Abcam Inc.), uncou- pling protein 3 (UCP3, 1:1000; ab3477, Abcam Inc.), tumor necrosis factor – alpha (TNF-a, 1:3500; ab9739, Abcam Inc.), heat shock protein 70 (Hsp70, 1:500; MAB3846, Millipore), Bcl-2- associated X protein (Bax, 1:3500; AB2915, Millipore), B-cell lymphoma 2 (Bcl-2, 1:500; ab32124, Abcam Inc.) and caspase 9 & cleaved caspase 9 (1:200; 9509, Cell Signalling Technology, Danvers, MA) overnight at 4uC or for 2 h at room temperature. Equal loading was ensured by ponceau staining as well as probing for GAPDH (1:500 000; MAB374, Millipore). The antigen- antibody complexes were detected by incubating the membrane with anti-mouse (1:10 000; AP160P, Millipore; COX-I, COX-IV, Hsp70, GAPDH) or anti-rabbit [(dilution 1:5000; NA934V, Amersham, GE Healthcare, Baie d’Urfe´, Quebec; CS, Cu/Zn SOD, MnSOD, UCP3), (1:5000; AP307P, Millipore; TNF-a, Bcl- 2, Bax) and (1:2000; 7074, Cell Signalling; caspase 9, cleaved caspase 9)] HRP conjugated secondary antibodies at room temperature for 1 h. Immunoreactive proteins were visualized with enhanced chemiluminescence (RPN2132, Amersham, GE Healthcare; COX-I, COX-IV, CS, Cu/Zn SOD, MnSOD, and UCP3; or WBKLSO500, Millipore; TNF-a, Bcl-2, Bax, Hsp70, caspase 9, and cleaved caspase 9), and exposed to Kodak autoradiographic film (COX-I, COX-IV, CS, Cu/Zn SOD, MnSOD, and UCP3) or Progene autoradiographic film (TNF-a, Bcl-2, Bax, Hsp70, caspase 9, cleaved caspase 9). Films were scanned and the protein intensity was normalized to GAPDH using Image J (v 1.37, NIH, USA) from the NIH. Statistical Analysis A three-way repeated measures ANOVA was used to determine significant differences in food intake, body weight, body condition, ability to move and PaGE, the between-subject factors being sex and diet (CR vs. AL), and the within-subject factor being time. This analysis was conducted on the 53 mice that were carried to endpoint. We conducted statistical analysis (3-way repeated measures ANOVA: 3 factors being sex, diet and time) and verified that the mice in the surgery group had outcome variables similar to the mice led to endpoint. No significant group effect was found for any of the following variables: body weight, body condition, ability to move, clinical onset and PaGE. A two-way ANOVA was used to determine significant differences in MDA, PC, CS, COX and catalase enzyme activity, and protein content of MnSOD, Cu/Zn-SOD, UCP3, Hsp70, TNF-a, Bax, Bcl-2, caspase 9, cleaved caspase 9, the factors being sex and diet (CR vs. AL). When significance occurred (P#0.05), a Tukey’s HSD post hoc test was used to determine the source of the difference. One-tailed tests were used to determine differences between the diets within each sex for MDA and PC because we hypothesized a priori that oxidative stress would be higher in CR vs. AL mice. We hypothesized a priori that MnSOD, Cu/Zn-SOD and UCP3 would also increase as a compensatory response to the heightened oxidative stress, thus a one-tailed test was employed for these outcome measures. A one-tailed test was also used for PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9386