Tgf βri

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.

Erythrocytes were lysed on ice using Mammalian Protein Extraction Reagent (CWBiotech, Beijing, China), and protein concentrations were determined using Varioskan Flash (Thermo, Waltham, MA, USA). Lysate supernatants were boiled for 10 min, separated on an 10% SDS-polyacrylamide gel, and then transferred to nitrocellulose membranes (Bio-sharp, Beijing, China). Membranes were blocked for 1 h in 5% skim milk in Tris-buffered saline with Tween (TBST) and were then incubated with antibodies against Band 3 (1:1000, Abcam, Cambridge, MA, USA), TGF-β RI (1:1000, Abcam, Cambridge, MA, USA), Act RII (1:1000, Abcam, Cambridge, MA, USA) and α-tubulin (1:2000, Abcam, Cambridge, MA, USA) overnight at 4 °C. Membranes were washed three times with TBST. Membranes were then incubated with peroxidase-conjugated immunoglobulin G antibody (1:5000; Jackson Immunoresearch, Lancaster, PA, USA) for 2 h at RT before washing with TBST. After adding developing liquid, chemiluminescent signals were detected using a Tanon-5200 system (YuanPingHao Biotech, Beijing, China).