Af1819

The ROS (#DRE901Mu, ShangHaiLianShuo Biological Co., Ltd.), SOD (R&D Systems, #DYC3419-2) and MDA (#EU2577, Wuhan Fine Biotech Co., Ltd.) levels in mice renal tissues and α-klotho (R&D Systems, #AF1819), TNF-α (R&D Systems, #MTA00B) and IL-6 (Sigma, #RAB0308-1KT) levels in mice serum were detected by ELISA kit. In addition, ROS (#NG-EA691, ShangHaiYuanmin Biotechnology Co., Ltd.), TNF-α (R&D Systems, #DTA00D) and IL-6 (Sigma, #RAB0306-1KT) levels in HRGECs cellular supernatant were detected by ELISA kit.

The interaction of Klotho or LRP6 and Wnt9a in HKC‐8 cells was determined by co‐immunoprecipitation, as previously reported (Zhou et al., 2013). Cells were transfected with Wnt9a expression vector (pFlag‐Wnt9a) for 24 hours, and then co‐treated with recombinant human Klotho (5334‐KL; R&D Systems) for 12 hours. Cell lysates were immunoprecipitated overnight at 4°C with an antibody against Wnt9a (ab125957; Abcam) using protein A/G plus agarose (sc‐2003; Santa Cruz Biotechnology). After washing three times, the precipitated complexes were immunoblotted with anti‐Wnt9a (ab125957; Abcam), anti‐Klotho (AF1819; R&D Systems), and anti‐LRP6 (ab134146; Abcam) antibodies. Cell lysates were also tested by an antibody against GAPDH (RM2001, Ray Antibody Biotech).

Paraffin‐embedded (3 μm thickness) kidney sections were prepared in a routine procedure. Periodic acid‐Schiff (PAS), and Sirius red staining (DC0040, Leagene Biotechnology, Beijing, China) were performed according to the manufacturer's instruction. Immunohistochemical staining was performed using routine protocol. Slides were incubated with antibodies against anti‐Klotho (AF1819; R&D Systems), anti‐fibronectin (F3648; Sigma), anti‐γH2AX (ab2893; Abcam), anti‐PGC‐1α (ab54481; Abcam), anti‐TOMM20 (ab186735; Abcam), Biotin‐sp‐conjugated affinipure donkey anti‐mouse, anti‐rabbit IgG or anti‐goat IgG (Jackson Immuno‐Research Laboratories, West Grove, PA). Images were taken by a microscope DP 27 CCD camera (Olympus, Japan).

For immunoblotting, lysates of cells and tissues were lyzed in RIPA buffer or NP-40 buffer (150 mM NaCl, 50 mM HEPES pH7.4, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 1% Nonidet P-40, 2 M Na ortho-vandate and 1 mM DTT supplied with protease inhibitors leupeptin 10 μg/L, aprotinin 10 μg/L, and PMSF 1 mM) were prepared and denatured. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose or PVDF, stained by Ponceau S, blocked in dried nonfat milk 5%-TBST or bovine serum albumin (A9647 Sigma-Aldrich) 3% in TBST before incubation with primary antibodies against Memo (1:2000, produced in-house (Haenzi et al. 2014 (link)) or 1:1000, HPA042603 Sigma-Aldrich), pERK (1:1000, sc-7383 Santa Cruz, Dallas TX, USA), tERK (1:1000, sc-93 Santa Cruz), actin (1:2000, A2066 Sigma-Aldrich), Rho-GDI1 (1:500, ABIN969501 Antibodies-online), Rac1 (1:500, ab33186 Abcam, Cambridge UK), RhoA (1:500, NBP2-22529 NovusBio) and Klotho (1:1000, AF1819 R&D Systems). Membranes were incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Milian Analytica, Rheinfelden Switzerland or ImmunoResearch, distributed through LubioScience GmbH, Zürich, Switzerland) and imaged using Fusion Solo (Witec AG, Sursee Switzerland) or a ChemiDoc XRS + System (BioRad). For reprobing, membranes were stripped using a low pH buffer (25 mM glycine–HCl, pH2, 0.4% (w/v) SDS).

Western blot analysis was performed as described previously (Li et al., 2019 (link)). Briefly, lysed samples (40 μg of protein) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, MA, United States). The membranes were incubated with the antibodies against α-klotho (AF1819, R&D, MN, United States), β-actin (#4970, CST, Beverly, MA, United States), and GAPDH (ab8245, Abcam, Cambridge, United Kingdom). The bands were visualized with enhanced chemiluminescence and quantified by densitometry. The levels of proteins were normalized according to the level of β-actin or GAPDH.