Concentrations of supernatants were determined by BCA assay (Thermo Scientific). Western blotting was performed as previously described [9]. Briefly, 25 g of each sample was loaded onto a 12 Monocrotaline dose Bis-Tris-polyacrylamide gel (Invitrogen) and transferred to nitrocellulose membranes. Blots were first probed with specific antibodies as follows: CTR1 (sc-66847, Santa Cruz Biotechnology Inc); CTR2 (sc104852, Santa Cruz Biotechnology Inc); MT1/2 (sc-11377, Santa Cruz Biotechnology Inc), and then incubated with an HRP-conjugated IgG, (dilution 1:10,000, Jackson Laboratories). Specific protein bands were detected by using an enhanced chemiluminescent substrate (ECL Plus, GE Healthcare, Buckinghamshire, UK) and imaged using an LAS 3000 image reader (Fuji Photo Film Co. Ltd, Tokyo, Japan). Ponceau-S staining was used as the reference for loading controls, and band intensities were measured using MultiGauge software v2.0 (Fuji Photo Film).Cu/Zn-SOD1 ELISACopper concentrations were determined PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 in dry ex vivo LV-tissue by using a reference method, PIXE coupled with RBS [57]. The calibration, measurements, and limits of detection were based on the areas of the K x-ray peaks as measured by the software package GUPIX Elemental, and concentrations were extracted from PIXE spectra using GUPIX software (University of Guelph, ON, Canada), as previously described [10,46].Measurement of mRNA by RT-qPCRHere, RT-qPCR was performed according to `The Minimum Information for Publication of Quantitative RealTime PCR Experiments (MIQE)’ guidelines [58]. Total RNA was extracted from rat LV tissues that had previously been preserved (RNAlater, Ambion), by using an RNeasy mini kit (Qiagen), and was reverse-transcribed into cDNA using a first-strand cDNA synthetase kitLV tissues were weighed and minced into small pieces before homogenization in phosphate-buffered saline (pH 7.2; 20 l/mg tissue) using a TissueLyser II (Qiagen). The resulting suspension was subjected to two freeze-thaw cycles and then centrifuged (5 min, 5000 ?g, 4 ). Thereafter, supernatants were removed and totalprotein concentrations determined by bicinchoninic acid (BCA) assay (Thermo Scientific). SOD1 concentrations were measured using an ELISA kit according to the manufacturer’s protocol (USCN Life Science; Wuhan, Hubei, PRC) by spectrophotometry (450 nm, microplate reader; SpectraMAX 340, Molecular Devices; Sunnyvale, CA), and the concentration of SOD1 (ng/mL) in the sample supernatants calculated by comparing the O.D. of the samples to a standard curve. Results were then converted to ng/mg total protein and expressed as concentrations relative to the corresponding control group.Zhang et al. Cardiovascular Diabetology 2014, 13:100 http://www.cardiab.com/content/13/1/Page 5 ofCu/Zn-SOD1 activity assayLV tissues were homogenized in ice-cold 0.1 M Tris/ HCl, pH 7.4 containing 0.5 Triton X-100, 5 mM mercaptoethanol and 0.1 mg/ml PMSF using a TissueLyser II (Qiagen). The crude tissue homogenates were then centrifuged (14,000 , 5 min, 4 ) and the supernatants collected: these contained total SOD activity from combined cytosolic and mitochondrial sources. Following incubation with 0.4 vol of a solution containing ethanol and chloroform (25:15 vol/vol) to inactivate the mitochondrial SOD2 [59], mixtures were centrifuged at 5000 ?g for 15 min and aliquots of the supernatant used to measure protein concentration by BCA assay, and Cu/ Zn-SOD activity using the Superoxide Dismutase Activity Assay kit (Abcam). The.