Ab181373

Cells were washed twice with ice-dole PBS and lysed in RIPA buffer (150 mM NaCl, 1% NP-40,,50 mM Tris/HCl, pH 8.0 and 10% glycerol) supplemented with 100 μg/ml phenylmethylsulfonyl fluoride (PMSF) for 10 min. Cell debris was removed by centrifugation at 12,000 rpm for 20 min at 4 °C. Protein concentrations were determined by using Thermo Pierce® BCA Protein Assay Kit. 20 μg total protein lysate was subjected to electrophoresis in denaturing 10% SDS-polyacrylamide gel, and then transferred to a membrane for subsequent blotting with antibodies. KL antibody was obtained from Abcam (ab181373). Flag antibody was purchased from Sigma (F1804). Rabbit monoclonal antibody against ERK1/2 (9101), and phosphor-ERK1/2 (4370) were purchased from Cell Signaling Technology. β-actin (60008–1-lg), HIF1α (20960–1-AP), HK2 (22029–1-AP), Glut1 (21829–1-AP), LDHA (19987–1-AP) antibodies that purchased from Proteintech.

Total proteins of frozen kidney tissues and harvested cells were extracted with radioimmunoprecipitation buffer (R0020, Solarbio) with a protease inhibitor cocktail (P6730, Solarbio). After quantification with a BCA protein assay kit (No. 23227, Thermo Fisher), protein (20 μg) was separated by 8% or 10% SDS-PAGE and then transferred to a PVDF membrane. The membranes were blocked in 5% non-fat milk at room temperature (RT) for 1 h and then incubated with primary antibodies overnight at 4°C. Primary antibodies used were shown below: anti-Klotho (ab181373, Abcam; sc-515942, Santa Cruz), anti-DNA methyltransferase (DNMT) 1 (#5032, CST), anti-DNMT3a (#3598, CST), anti-DNMT3b (#67259, CST), anti-p-STAT3 (ab76315, Abcam), anti-α-smooth muscle actin (α-SMA; #14968, CST), anti-E-cadherin (E-cad; #14472, CST), and anti-GAPDH (ab181603, Abcam). The next day, membranes were incubated with HRP-labeled secondary antibodies (sc-2357 & sc-2005, Santa Cruz) for 1 h at RT. Immunoblot signals were detected and then analyzed by Image J software with normalization to GAPDH.

The rabbit polyclonal α-Klotho antibody (catalog no. Ab69208 and Ab181373; Abcam) and rabbit polyclonal isotype antibody (catalog no. Ab27478) were used at a concentration of 1:100 to 1:250 for IHC staining and control experiments on formalin-fixed and paraffin-embedded, glass-mounted sections. Please see Supplemental Data for details of the IHC protocol.
Assessment of α-Klotho distribution along the nephron was done morphologically. Determination was limited to proximal tubular and distal tubular (distal tubule and collecting duct) nephron segments. Consecutive section staining with hematoxylin and eosin was also used.

Two lung adenocarcinoma tissues and paired paracarcinoma were used for immunohistochemical staining (IHC). In brief, paraffin-embedded tissue sections were deparaffinized, rehydrated, and pretreated for epitope retrieval. After blocked with 5% goat serum for an hour, the sections were incubated with appropriate primary antibodies overnight at 4 degrees. The primary antibodies used were from Abcam/Santa Cruz/Servicebio/Invitrogen: ADIPOR1 (1:500, ab126611, Abcam), ARRB1 (1:200, ab32099, Abcam), S100A12 (1:20, sc-101347, Santa Cruz), CD1b (1:200, Abcam, ab173576), HAMP (1:50, sc-101347, Santa Cruz), HMOX1 (1:1000, GB11845, Servicebio), KL (1:200, ab181373, Abcam), S100A7 (1:20, sc-52948, Santa Cruz), S100A2 (1:2000, GB111077, Servicebio), VEGFA (1:20, sc-7269, Santa Cruz), VIPR1 (1:50, Invitrogen, PA3-113), and TUBB3 (1:50, sc-80016, Santa Cruz). Following incubation with an HRP-conjugated secondary antibody (1:300, K8002, Dako), the stained sections were reacted with 3,3’-diaminobenzidine and counterstained with hematoxylin.

Paraffin-embedded aorta was cut into 4 μm thick sections. The slides were incubated with sodium citrate buffer for 10 min in a pressure cooker for antigen retrieval. After the incubation with blocking solution of 5% goat serum in TBST at room temperature for 10 min, the sections were incubated with primary antibodies against Runx2 (1:50 diluted with TBST buffer, SC-101145, Satan Cruz Biotech., USA) or Klotho (1:50 diluted with TBST buffer, ab181373, Abcam, UK) overnight at 4°C followed by the incubation with appropriate secondary antibodies conjugated with biotin for 15 min at room temperature. The sections were then incubated with HRP labeled streptavidin for 15 min at room temperature and stained with DAB solution. The nuclei were counterstained with hematoxylin solution. The images were acquired by a Leica DM4B fluorescence microscope and analyzed with ImageJ software. The results were expressed as a percentage of positive stained areas with total selected areas.

A 10 mM stock solution of EZH2 inhibitor (HY-13500, MedChemExpress Bio-Technology, Shanghai, China) was purchased and stored at −80 °C. The dilutions for the working solution did not exceed 0.1% DMSO in the medium. Dulbecco’s modified Eagle’s medium (DMEM F12/1:1), fetal bovine serum (FBS), and trypsin/EDTA were purchased from HyClone (Logan, UT, USA). Nile red (N8440) and Oil red O stains (G1262) were purchased from Solarbio Company. Klotho siRNA (sc-43883) was purchased from Santa Cruz Biotechnology. A tissue triglyceride assay kit (E1013) was purchased from Pplygen Company (Beijing, China). The primary antibodies were as follows: anti-EZH2 (ab191080, Abcam, Cambridge, MA, USA) for WB, as well as anti-E-cadherin (ab231303, Abcam, USA), anti-N-cadherin (ab76011, Abcam, USA), anti-vimentin (ab92547, Abcam, USA), anti-FN (ab2413, Abcam, USA), anti-klotho (ab181373, Abcam, USA) and anti-GAPDH antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by Beyotime Biotechnology (Shanghai, China). All other chemicals were of reagent grade and endotoxin free.

The tissues and cells were lysed by using the cold radioimmunoprecipitation (RIPA) buffer (Bio-Rad, CA, USA) on ice, and the cell lysates were heated for 5 min at 95 ℃ for protein denaturation, which were further subjected to 10% SDS-PAGE to separate the proteins according to their molecular weight. The target proteins were then transferred from the gels to PVDF membranes (Millipore, USA), and the proteins-loaded PVDF membranes were blocked with 5% non-fat milk for 40 min at room temperature. Then membranes were subsequently incubated with the primary antibodies against Klotho (1:1500, #ab181373, Abcam, UK), GAPDH (1:2000, #ab8245, Abcam, UK), p16 (1:1500, #ab51243, Abcam, UK), p21 (1:1500, #ab109520, Abcam, UK), Nrf2 (1:2000, #ab62352, Abcam, UK), SOD2 (1:1500, #ab68155, Abcam, UK) and NQO1 (1:2000, #ab80588, Abcam, UK) at 4℃ overnight. The antibodies were washed off and incubated with the horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, USA) for 1 h at 37 ℃. The ECL system (ThermoFisher Scientific, USA) was used to visualize the protein bands, which were further analyzed by using the Image J software.