4.5. Mitochondria Isolation and Western Blotting

Mitochondria were isolated from kidneys of OXYS rats kept on the AL or CR diet. The rat kidney mitochondria for western blotting were isolated by differential centrifugation in the medium containing 0.25 M sucrose, 10 mM HEPES, 1 mM EDTA, 0.1% BSA, pH 7.4 [100]. All further steps correspond to those for western blotting of kidney homogenates. Total protein loading was estimated by Stain-Free imaging technique according to the manufacturer’s instructions (#1610185, BioRad, USA). Membranes were incubated with primary antibodies: anti-Bcl-X 1:1000 rabbit (#2764, Cell Signaling, USA), anti-COX IV 1:1000 mouse (#A21348, Invitrogen, USA), anti-PINK-1 1:1000 rabbit (ab23707, Abcam, UK), anti-ubiquitin 1:1000 rabbit (ab7780, Abcam, UK), anti-Fis1 1:1000 rabbit (ab71498, Abcam, UK), anti-Drp1 1:1000 mouse (611739, BD Biosciences, Franklin Lakes, NJ, USA), anti-acetylated-Lysine 1:1000 rabbit (#9441, Cell Signaling, USA), anti-SIRT3 1:1000 rabbit (#2627, Cell Signaling, USA). Similarly, as for kidney homogenates, membranes were then incubated with secondary antibodies: anti-rabbit IgG or anti-mouse IgG conjugated with horseradish peroxidase 1:7500 (Jackson ImmunoResearch, USA) and probed with Advansta Western Bright ECL kit (Advansta, USA). The concentration of total mitochondrial protein was measured with bicinchoninic acid assay (Sigma, USA). Carbonylated proteins were detected using the OxyBlot kit according to the manufacturer’s instructions (S7150 OxyBlot Protein Oxidation Detection Kit, Millipore, USA).