The blots were probed with the appropriate antibodies to assess the protein level of the HSP70 (Stressgen, Victoria, BC, Canada; Ref SPA810 diluted 1:3000 and 1:500 for exercised and sedentary rats, respectively), glutamine synthetase (GS) (Abcam, Cambridge, Ref. ab64613 diluted 1:1000) and tubulin (Abcam, Cambridge, Ref. ab44928 diluted 1:1000). The appropriate secondary mouse antibody conjugated to peroxidase and the BM chemiluminescence blotting system (Abcam) were

used for detection. The bands were visualised using a GE ImageQuant, model Fasudil nmr LAS 4000 instrument. Specific protein bands present in the blots were quantified using the digital program ImageJ (v. 1.44 for Windows). The protein sources were hydrolysed at 110 °C in 6 M HCl for 24 h. The hydrolysed samples (wet basis) were then diluted in deionised water; α-aminobutyric acid was added as the internal standard (Sigma–Aldrich Corp., St Louis, MO), and the amino acids were derivatised with phenylisothiocyanate. The PTH-derivatives were chromatographed using a Luna C-18, 100 Ǻ; 5 μm, 250 × 4.6 mm column (Phenomenex, Torrance, CA), at 50 °C. The plasma free PLX3397 mw amino acids were extracted with trichloroacetic acid and then derivatised and chromatographed as described above. Blood samples were collected in Vacutainers, kept at 4 °C,

and centrifuged at 3000g (4 °C, 15 min) to obtain the serum and plasma. The sera were assessed for uric acid, creatine kinase (CK), lactate dehydrogenase (LDH), total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase CYTH4 (ALT), creatinine and urea using spectrophotometric (Beckman-Coulter DU 640, Palo Alto, CA) determinations employing Laborlab

kits (São Paulo, Brazil). Glucose in the blood was measured using an Accu-Chek Active glucometer (Roche Diagnostics, Mannheim, Germany). Skin temperature was measured both before and after the last exercise session with an infrared thermometer ( Luong & Carrive, 2012) (Geratherm Medical Diagnostic Systems, Geschwenda, Germany). Corticosterone (CORT) was determined using an enzyme immunoassay kit (Assay Designs – Stressgen, catalogue 900.097; Enzo Life Sciences, Exeter, UK). Samples of the gastrocnemius muscle (150–200 mg) were mixed and homogenised in 3 mL of 50 mM phosphate buffer, pH 7.4, containing 0.1% digitonin, and a cocktail of antiproteases (40 μg/mL phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, 7 μg/mL, pepstatin, 5 μg/mL aprotinin and 1 mM EDTA). The plasma (100 μL) was directly homogenised in 2,4-dinitrophenylhydrazine (DNPH). The carbonyl groups reacted with the DNPH, and after successive deproteinisation and dissolution procedures in guanidine hydrochloride, the spectra from 355 to 390 nm were read in a spectrophotometer (Epoch micro-plate reader; BioTek, Instruments, Inc., Winooski, VT) according to a previously described method (Reznick & Packer 1994).