Anti proliferating cell nuclear antigen pcna

The tissues were ground in liquid nitrogen into a powder using a mortar and a pestle and subjected to radioimmunoprecipitation assay buffer (Sigma-Aldrich) for lysis; the immunoblots were performed using methods described previously20 (link). The primary antibodies included anti-Sirt1 (1:250 in glomerular cytoplasmic and nuclear fraction samples, and 1:1000 in others; Sigma-Aldrich), anti-β-actin (1:10000; Cell Signaling Technology, Danvers, MA), anti-proliferating cell nuclear antigen (PCNA), anti-Lamin A/C (both 1:1000; GeneTex Inc., Irvine, CA), anti-ghrelin (1:500; Santa Cruz Biotechnology Inc.), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:2000; GenScript, Piscataway, NJ). Horseradish peroxidase-conjugated secondary antibodies were used, and the hybridization signals were amplified by Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences, Piscataway, NJ). Densitometry was performed using ImageJ v1.48 (National Institutes of Health, USA).

Total proteins were extracted from cultured cells or homogenized renal tissues using radioimmunoprecipitation assay (RIPA) buffer (Sigma), and western blotting analysis was performed as described previously18 (link). The following primary antibodies were used: rabbit polyclonal anti-Sirt1 (1:800; Sigma), anti-cleaved caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA), anti-nitrotyrosine (1:1000; Santa Cruz), anti-AT1R (1:500; Millipore), anti-AT2R (1:500; Millipore), anti-NF-κB (p65) (1:5000; Enzo Life Science, Farmingdale, NY, USA), anti-acetyl (K310)-NF-kB p65 (1:1000; Abcam, Cambridge, MA), anti-phospho (Ser32)-IκBα (1:1000; Cell Signaling), anti-connective tissue growth factor (CTGF; 1:1000; Peprotech, Rocky Hill, NJ, USA), hamster monoclonal anti-monocyte chemotactic protein 1 (MCP-1; 1:500; Abcam), mouse monoclonal anti-fibronectin (1:400; Abcam), anti-GAPDH (1:2000; GenScript, Piscataway, NJ, USA), and anti-proliferating cell nuclear antigen (PCNA, 1:1000; GeneTex Inc., Irvine, CA). The secondary horseradish peroxidase (HRP)-conjugated antibodies included goat anti-rabbit IgG (Santa Cruz Biotechnology), goat anti-mouse IgG (PerkinElmer, Waltham, MA, USA), and goat anti-hamster IgG (Invitrogen) diluted 1:5000. Specific signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore).