Anti p rr

NRK52Ecells were cultured on15 × 15-mm coverslips in 35-mm plates and treated with or without prorenin (100 pmol/L, 48 h). Indirect immunofluorescence staining was performed as described in our previous study17 (link). Cells were stained using anti-V-ATPase E subunit (1:25, Abcam, Cambridge, UK), anti-(P) RR (1:50, Abcam, Cambridge, UK), or anti-V-ATPase B2 (1:50, Santa Cruz, CA) antibodies or double-stained with anti-(P) RR and anti-V-ATPase B2 antibodies, followed by staining with anti-mouse Alexa Fluor 546, or anti-mouse Alexa Fluor 488, or anti-rabbit Alexa Fluor 488antibodies (1:1,000, Invitrogen). Cells were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (1:200, Invitrogen) for 7 min to identify nuclei. Coverslips were secured using Fluoro-gel with Anti-Fading Agent (Electron Microscopy Sciences, Hatfield, PA). Slides were viewed, and images were collected and analyzed using a laser-scanning confocal microscope and LSM5 image browser software (LSM 510META, Zeiss, Oberkochen, Germany).

Western blot analysis was performed as we described previously. In brief, homogenates from cultured podocytes were prepared using sucrose buffer containing protease inhibitor. After boiled for 5 min at 95°C in a 5× loading buffer, 20 µg of total proteins were subjected to SDS-PAGE, transferred onto a PVDF membrane and blocked by solution with dry milk. Then, the membrane was probed with primary antibodies of anti-PRR (1∶1000, Abcam), anti-Wnt3a (1∶1000, Abcam), anti-β-catenin (1∶500, R&D system), anti-snail (1∶500, Novus), anti-nephrin (1∶100, Santa Cruz), anti-podocin (1∶1000, sigma) or anti-β-actin (1∶5000, Santa Cruz) overnight at 4°C followed by incubation with horseradish peroxidase-labeled IgG (1∶5000). The immunoreactive bands were detected by chemiluminescence methods and visualized on Kodak Omat X-ray films. Densitometric analysis of the images obtained from X-ray films was performed using the Image J software (NIH, Bethesda, MD, USA).

Total protein was extracted from myocardial tissue and fibroblasts. The total protein concentration was determined, and the proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. After blocking nonspecific binding in 5% skim milk for 1.5 h, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-pAMPK (1:1000, Affinity, United States), anti-AMPK (1:1000, Affinity, United States), anti-PRR (1:1000, Abcam, United Kingdom), anti-GAPDH (1:1000, Abcam, United Kingdom), and anti-YAP (1:1000, Cell Signaling Technology, United States). Then, horseradish peroxidase-conjugated secondary antibodies (1:5000, ZSGB-bio, China, ZB-2301) were used to bind to the specific antigen-antibody complexes. The densitometry of the bands was performed with ImageJ (National Institutes of Health, Bethesda, MD, United States).