Western blot analysis

For protein preparation, cells and kidney tissue were suspended in radioimmunoprecipitation assay buffer. The cells were then lysed on ice for 30 minutes, and the cell lysate was collected by centrifugation at 15,000 ×g for 10 minutes. Protein quantification was performed using a Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA, USA). Then, 30 µg of proteins were electrophoresed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk in Tris-buffered saline containing Tween 20 (0.1%) for 1 hour, the membrane was incubated with anti-type I collagen (1:1,000; Abcam), anti-fibronectin (1:1,000; BD Biosciences), anti-phospho-Smad3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), and anti-TGF-β (1:1,000; Cell Signaling Technology) polyclonal antibodies at 4℃ with gentle shaking overnight. Antibodies were detected by horseradish peroxidase-linked secondary antibody (Santa Cruz) using the enhanced chemiluminescence Western Blotting Detection System, in accordance to the manufacturer's instructions (Amersham, Buckinghamshire, UK). The membrane was reblotted with anti-β-tubulin antibody to verify equal loading of the protein in each lane. Densitometric measurements of the bands were made using the digitalized scientific program UN-SCAN-IT (Silk Scientific Corp., Orem, UT, USA).