Core 2

Mitochondria isolation from kidneys of Sham and CLP groups was performed using sucrose containing buffer as described previously [18 (link),19 (link)]. Mitochondrial complexes were extracted from the isolated mitochondria (250 μg) using 10% n-dodecyl-β-D-maltoside and 0.5 M aminocaproic acid (detergent/protein ratio, 2.5 g/g). The mitochondrial extracts (40 μg) were then resolved in a BN-PAGE gel [19 (link),20 (link)] followed by western blotting with 4-HNE (Abcam, 1:500) and Core-2 (Abcam,#ab14745;1:1000).

Skeletal muscle and heart tissues were resuspended in lysis buffer (1% Tx-100, 10 mM Tris-HCI pH 7.6, 150 mM NaCl, 1 mM EDTA) with protease inhibitor and phosphatases (Roche Diagnostic) and then were centrifuged at 11,000× g for 15 min (4 °C). Protein concentration was quantified with Micro BCA Kit (Thermo Fisher Scientific, Waltham, MA, USA). Samples were incubated for 5 min in TC 4X buffer (β-Mercaptoethanol) at 95 °C and 30 µg of protein extracts were separated by SDS-PAGE electrophoresis in 12.5% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (GH Healthcare, Chicago, CA, USA). The following antibodies were used for inmunodetection: NDUFB11 (Abcam, 1:1000, Cambridge, UK), NDUFA9 (Abcam, 1:1000), NDUFV1 (Santa Cruz Biotechnology, 1:1000, Dallas, TX, USA), NDUFB8 (Abcam, 1:500), Core2 (Abcam, 1:2000) and COXII (Abcam, 1:1000). SDHA (Abcam, 1:10,000) was used as loading control. Secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technologies, Danvers, MA, USA) were used to detect the primary antibodies and the reactions were developed with ECL Prime Western Blotting Detection Reagent (GE Healthcare, Amersham, UK) in a ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA). The optical densities of the immunoreactive bands were measured using NIH ImageJ software v1.8 (Wayne Rasband, NIH, Bethesda, MD, USA).