Monoclonal cyp a

Immunohistochemistry was performed as described previously [27 (link)]. The following primary antibodies were used: monoclonal anti-CAV-1 (1:250; Epitomics, Burlingame, CA, USA), monoclonal CyP A (1:150; GeneTex, Irvine, CA, USA), monoclonal anti-peroxisome proliferator-activated receptor-γcoactive 1α (PGC-1α; 1:250; Abcam), polyclonal anti-nuclear respiratory factor-1 (NRF-1; 3 μg/mL; GeneTex), polyclonal anti-superoxide dismutase 2 (SOD2; 1:1000, Novus, Littleton, CO, USA), and polyclonal anti-catalase (1:1000; Abcam). monoclonal anti-nuclear factor E2-related factor 2 (Nrf2; 1:250; GeneTex), polyclonal anti-Kelch-like ECH- associated protein 1 (Keap1; 1:150; Bioss, Woburn, MA, USA), and polyclonal anti- glutamate-cysteine ligase catalytic subunit (GCLC; 1:100; GeneTex). Nuclei were counterstained with hematoxylin and photographed using an Olympus BX61 microscope (Tokyo, Japan). For animal kidney tissue, positive cells were quantified in five fields per animal at 400×, with six rabbits for each group per time point (Image-Pro Plus 4.5).

Tissue lysate was harvested in a lysis buffer (25 mM bicine, 150 mM sodium chloride, pH 7.6; Pierce), homogenized, and centrifuged for 20 min at 12 000 rpm at 4°C. Nuclear and cytoplasmic fractions were prepared from renal tissue using a Nuclear Protein Isolation-Translocation Assay Kit (FIVEphoton Biochemicals, San Diego, CA, USA) according to manufacturer instructions. The protein concentration was detected using a BCA protein assay kit (Pierce BCA assay, Thermo Scientific, Rockford, IL). Proteins (30 or 50 μg) were separated using 8% –12% SDS-PAGE and then transferred to PVDF membranes (Pall Corporation). Blots were then probed with monoclonal anti-CAV-1 (1:1000; Epitomics), monoclonal Cyp A (1:1000; GeneTex), monoclonal anti-PGC-1α (1 μg/mL; Abcam), polyclonal anti-NRF-1 (1:1000; GeneTex), polyclonal anti-SOD2 (1:1000, Novus), polyclonal anti-catalase (1:1000; Abcam), MitoProfile Total OXPHOS rodent antibody cocktail (1:800; MitoSciences, Eugene, OR), monoclonal anti-Nrf2 (1:800; GeneTex), polyclonal anti-Keap1 (1:1000; Bioss), polyclonal anti-GCLC (1:800; GeneTex), polyclonal anti-Lamin B1 (1:1000; Abcam), mouse anti-GAPDH (1:1000; Abcam), and mouse anti-β-actin (1:10000; Millipore, Billerica, MA, USA). Signals were obtained using an enhanced chemiluminescence kit (Millipore), and densitometry was performed using Fusion-Capt software (Vilber Lourmat, Fusion FX7, France).