Extraction and Separation of Proteins

Collected cells or renal tissues were lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) on ice. The cell or tissue lysing reagents were collected, centrifuged at 14,000× g for 10 min at 4 °C, and mixed with 5 × SDS-PAGE Sample Loading Buffer (Beyotime Biotechnology, Shanghai, China). Proteins were separated by 10~15% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) microporous membranes as described [43 (link)]. Membranes were subsequently developed with ECL reagent, and chemiluminescent signals were acquired using Tanon 5200 ChemiDoc imager (Shanghai Tianeng Technology Co., Ltd., Shanghai, China).