Monoclonal anti pgc 1α

Tissue lysate was harvested in 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. The protein concentration was detected using a BCA protein assay kit (Pierce BCA assay, Thermo Scientific, Rockford, IL). Protein (30 µg) was separated by 10% or 12% SDS-PAGE and then transferred to PVDF membranes (Pall Corporation). Blots were then probed with monoclonal anti-CAV-1 (1∶1000; Epitomics, Burlingame, CA, USA), monoclonal anti-PGC-1α (1 µg/ml; Abcam, Cambridge, MA, USA), polyclonal anti-NRF-1 (1∶1000; GeneTex, Irvine, CA, USA), polyclonal anti-SOD2 (1∶1000, Novus, Littleton, CO, USA), polyclonal anti-catalase (1∶1000; Abcam), MitoProfile Total OXPHOS rodent antibody cocktail (1∶800; MitoSciences, Eugene, OR), and mouse anti-β-actin (1∶10,000; Millipore). Signals were obtained using an enhanced chemiluminescence kit (Millipore, Billerica, Massachusetts, USA) and densitometry was performed using Fusion-Capt software (Vilber Lourmat, Fusion FX7, France).

Immunohistochemistry was performed as described previously [37] (link). The following primary antibodies were used: monoclonal anti-CAV-1 (1∶250; Epitomics, Burlingame, CA, USA), monoclonal anti-PGC-1α (1∶250; Abcam, Cambridge, MA, USA), monoclonal anti-reactive oxygen species modulator 1 (ROMO1) (1∶150, OriGene, Rockville, MD, USA), which has been reported to generate ROS in mitochondria [39] (link), polyclonal anti-NRF-1 (3 µg/ml; GeneTex, Irvine, CA, USA), , polyclonal anti-SOD2 (1∶1000, Novus, Littleton, CO, USA), and polyclonal anti-catalase (1∶1000; Abcam). Nuclei were counterstained with haematoxylin and photographed using an Olympus BX61 microscope (Tokyo, Japan). For animal liver tissues, positive cells were quantified in 5 fields per animal at ×400, N = 6 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).