2.7. Western Blot Analysis for TGF-β1, Smad3/7, p-Smad3, and SIP1

Renal tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (200 mg tumor tissue/1 ml RIPA, Beyotime Biotechnology, Shanghai, China) for 30 min on ice, and then centrifuged at 12,000 rpm for 5 min, supernatants were collected. The protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Solarbio, Beijing, China). Protein samples were separated by electrophoresis on 5% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Blocked in 5% (W/V) nonfat milk for 1.5 h, the membranes were incubated with the primary antibody against TGF-β1 (Abcam, Cambridge, UK), Smad3 (Affinity Biosciences, Ohio, USA), Smad7 (Affinity Biosciences, Ohio, USA), SIP1 (Abcam, Cambridge, UK), p-Smad3 (Cell Signaling Technology, Massachusetts, USA), and GAPDH (Proteintech, Illinois, USA) at 4°C overnight. After washing three times in TBST, the membrane was treated with horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling Technology, Massachusetts, USA) for 1 h and then washed three times in TBST. Density of the corresponding bands was measured by using chemiluminescence detection reagents (Solarbio, Beijing, China) and analyzed with Quantity One software.