Hpa003156

Protein samples were fractionated on SDS-PAGE (30 μg/well) and transferred to a nitrocellulose membrane (Millipore). Immunoblots were incubated overnight at 4°C with primary antibodies including anti-ACE (1:1,000, GTX100923, GeneTex, United States), anti-AGT (1:1,000, GTX103824, GeneTex, United States), anti-renin (1:1,000, sc-133145, Santa, United States), anti-PRR (1:1,000, HPA003156, Sigma, United States), anti-Npt2a (1:1,000, A6742, Abclonal, China), anti-Npt2c (1:1,000, ab155986, Abcam, United Kingdom) or anti-β-actin (1:10,000, A1978, Sigma, United States) antibody in 1.5% (w/v) bovine serum albumin (BSA, Sigma, United States) in a TBS-T buffer [150 mM NaCl, 10 mM Tris (pH 7.4/HCl), 0.2% (v/v) Tween-20]. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000, Thermo Fisher Scientific™ Pierce™). Specific signal was visualized by ECL kit (Thermo Fisher Scientific™ Pierce™). The protein bands were detected using Amersham Imager 600 and quantified by Image Pro Plus version 6.0 software (Molecular Dynamics).

Western blot analysis was performed as described previously^{8 (link)}. In brief, whole kidney homogenates were lysed in the presence of protease inhibitors. Clear protein extracts were obtained by centrifugation at 12,000 g for 10 min. 20 μg proteins were transferred to subjected to 4%-12% gradient SDS-PAGE, transferred onto a PVDF membrane filters (Bio Rad). PVDF membranes were blocked with 5% dry milk for 1 h. Membranes were incubated in the primary overnight at 4 °C. The following antibodies used in the study are: anti-PRR (1:1000, HPA003156, Sigma), anti-NOX4 (1:1000, ab133303, Abcam), anti-SOD2 (1:1000, ab13533, Abcam), anti-UCP2 (1:1000, ab203244, Abcam), anti-Caspase 3 (1:1000, CST #9665, Cell signaling), anti-pNF-κB Ser536 (1:1000, ab86299, Abcam), anti- tNF-κB (1:1000, ab16502, Abcam), anti-collagen IV (1:1000, ab6586; Abcam), anti-fibronectin (1:1000, ab2413; Abcam), anti-sirt1 (1:200, sc-15404, Santa Cruz), anti-pFOXO3a ser318/312 (1:1000, CST #9465, Cell signaling), anit-FOXO3a (1:200, sc-48348, Santa Cruz), and anti-β-actin (1:5000, sc-47778, Santa Cruz). This was followed by incubation with the corresponding secondary antibody (horseradish peroxidase-labeled IgG, 1:1000). β-actin was used to normalize per total amount of loaded proteins. Chemiluminescence blot images were captured by the UVP imaging and analyzed by using Image J software (NIH, Bethesda, MD).

Rabbit polyclonal antibody specific for PRR [ref. HPA003156; Sigma-Aldrich (Saint Louis, MO, USA) at 1/50 dilution] was used for the immunostaining of formalin-fixed and paraffin-embedded tumour tissues. The antibody’s specificity was tested previously [27 (link)] and the immunostaining process was performed following routine methods in an automatic immunostainer (Dako Autostainer Plus, Dako-Agilent, Santa Clara, California, USA). Briefly, antigen retrieval was carried out in a low-pH buffer (K8005, Dako, Santa Clara, California, USA) for 20 min at 95 °C. The samples were incubated with the primary antibody for 50 min at room temperature. Then, the primary antibody was washed and samples were incubated for 20 min with secondary anti-rabbit antibody (K8021, Dako). EnVision-Flex detection system together with an HRP-enzyme-labelled polymer (SM802, Dako) was employed. A positive reaction was visualized with diaminobenzydine (DAB) solution (DM827, Dako), followed by counterstaining with haematoxylin (K8008, Dako).
Slides were reviewed under light microscopy for staining evaluation. Two observers independently evaluated the slides and, in the event of discrepancies, samples were re-evaluated to arrive at a final conclusion. As previously reported [27 (link)], staining patterns were scored as negative, weak and intense cytoplasmic and membranous staining.