Anti klotho

Total proteins were extracted from the kidneys or HK‐2 cells and the concentration was measured by BCA protein assay kit (Beyotime, Shanghai, China). Western blot assay was performed with the standard method. Briefly, proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). PVDF membrane (Millipore, Bedford, MA, USA) was used to electro‐transfer. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with primary antibodies: anti‐Klotho (1:500, Abcam, #203576), TGF‐β1 (1:1000, Abcam, #92486), TGFβ‐RI (1:800, Abcam, #31013), TGFβ‐RII (1:1000, Abcam #186838), α‐SMA (1:1000, CST, #56856), Collagen I (1:2000, Abcam, #34710), Collagen IV (1:1500, Abcam, #6586), Smad 2/3 (1:1000, Santa Cruz, #398844), p‐Smad 2/3 (1:800, Abcam, #63399), Smad 4 (1:1000, CST, #38454) and Smad 7 (1:1000, Santa Cruz, #365846), followed by incubation with secondary antibody at room temperature for 1 hour. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as the internal control. Protein bands were visualized by enhanced chemiluminescence (ECL) reagent and exposed using BioImaging Systems (UVP, Upland, CA, USA). The relative protein levels were quantified using the Image J software (National Institutes of Health, Montgomery, MD, USA). All the assays were performed in triplicate.

Kidney and HK‐2 cells protein were extracted using RIPA buffer including protease inhibitor and PMSF (Heart, China). Samples protein concentration was measured by BCA Protein Quantitative Kit (Heart, China) and then adjusted to equal amounts for running western blot. Antibodies: anti‐p16 (1:1000; Abcam, England), anti‐p21 (1:1000; Abcam, England), anti‐p53 (1:1000; Abcam, England), anti‐Klotho (1:1000; Abcam, England), anti‐NLRP3 (1:1000; Cell Signaling Technology, USA), anti‐eNOS (1:1000; Cell Signaling Technology, USA), anti‐TGF‐β (1:1000; Abcam, England), anti‐IL‐6 (1:1000; Cell Signaling Technology, USA), anti‐IL‐1β (1:1000; Cell Signaling Technology, USA), anti‐Smad3 (1:1000; Proteintech, China), anti‐LTBP2 (1:1000; Santa cruz, USA), anti‐Tubulin (1:3000; Proteintech, China) and anti‐Gapdh (1:3000; Proteintech, China) were applied. Membranes were detected by chemiluminescence gel imaging system (FluorChem E; ProteinSimple, USA). Quantification of protein abundance was performed by Image J software 1.8.0.

Cell culture reagents and other general biochemical reagents including CaCl₂ and β-GP were purchased from Sigma–Aldrich (London, U.K.). Penicillin and streptomycin were from Fisher Scientific (Loughborough, U.K.), and FBS from Gibco (Scotland, U.K.). The bicinchoninic acid assay (BCA) protein quantification kit was from Thermo Scientific (Basingstoke, U.K.) and the DICA-500 Ca²⁺ assay kit was from Universal Biologicals (Cambridge, U.K.). Anti-Klotho, anti-RUNX2, anti-SM22α, and horseradish peroxidase (HRP)–conjugated secondary antibody (anti-rabbit) were from Abcam (Cambridge, MA). Anti-biotin was from Cell Signalling (Boston, MA) and HRP–conjugated anti-β actin was from Sigma (St. Louis, MO).