Collagen 4

GECs were lysed in RIPA buffer [50 mM Tris·HCl, pH 7.3; 150 mM NaCl; 0.1 mM EDTA; 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; 0.2% NaF; and 100 µM Na₃VO₄] supplemented with cOmplete protease inhibitors (Roche Applied Science, Indianapolis, IN). The kidney homogenate was centrifuged at 12,000 g for 30 min at 4 °C, and the protein concentration of the supernatant was determined by the Bradford method. Different amounts of extracted protein were separated by SDS-PAGE and transferred onto Immobilon-FL 0.4-μm polyvinylidene difluoride membranes (Millipore). Tris-buffered saline containing 0.1% Tween 20 was used as the wash buffer. After the membranes were treated with primary antibodies, anti-rabbit (1:5,000; Cell Signaling Technology, Danvers, MA) and anti-mouse (1:6,000 for β-actin; Cell Signaling Technology) secondary antibodies were used as appropriate. Detection of labeled proteins was performed with an enhanced chemiluminescence system (ECLTM PRN 2106; Amersham Pharmacia Biotech, Buckinghamshire, UK). The band intensities were analyzed using a gel documentation system (Bio-Rad Gel Doc 1000 and Multi-Analyst version 1.1).
Immunoblotting was performed using primary antibodies against collagen-1 (Abcam), collagen-4 (Abcam), fibronectin (Santa Cruz Biotechnology, Dallas, TX), αSMA (Abcam), and β-actin (Cell Signaling Technology).

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.

The Click-iT® EdU Alexa Fluor® 488 Imaging Kit and Alexa Fluor® 594 phalloidin were from Invitrogen. Matrigel (lrECM) and Type I collagen were from BD Bioscience. ShP4HA2 plasmids were purchased from Sigma. 1, 4-DPCA was purchased from Cayman Chemical. Masson’s trichrome stain kit was purchased from Polysciences, Inc. The following antibodies were obtained as indicated: integrin α6 (Millipore); collagen I (Abcam); collagen IV (Abcam); P4HA2 (Santa Cruz); tubulin (Millipore).