Amersham imager 600 system

Cells and kidney samples were homogenized and ruptured using ice-cold radio-immunoprecipitation assay (RIPA) buffer containing fresh protease and phosphatase inhibitors (Beyotime, China), and the protein concentration was measured using the bicinchoninic acid (BCA) assay (Beyotime, China). After denaturation, protein samples were subjected to SDS-PAGE, followed by immunoblotting with antibodies. Blots were visualized with an Enhanced ECL Chemiluminescent Substrate Kit (Yeasen, 36222ES76) and the Amersham Imager 600 System.

Total protein was collected from cells using RIPA buffer (Solarbio Life Science, Beijing, China) containing protease inhibitors (Beyotime Institute of Biotechnology, Jiangsu, China). The concentration of the extracted protein samples was then quantified using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). The proteins (30 µg) were subjected to Western blotting with the Bio–Rad Bis-Tris gel system. The polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA) were incubated with primary antibodies against RhoA (BM4479; Boster Bio, Pleasanton, CA, USA), ROCK1 (BM4203; Boster Bio), ROCK2 (BM5257; Boster Bio), pMLC (no. AF8010; Affinity Biosciences, Changzhou, China), MLC (DF7911; Affinity Biosciences) and Cx43 (ER1802-88; HuaBio, Hangzhou, China) diluted 1:1000 at 4°C overnight. Later, the PVDF membranes were probed with secondary antibody conjugated to horseradish peroxidase (Boster) for one hour. After rinsing, the PVDF membranes were placed in an Amersham Imager 600 system (Amersham, Piscataway, NJ, USA) and covered with BeyoECL Star reagent (Beyotime Institute of Biotechnology). The signals were captured with the fluorescence imaging system (Amersham Imager 600 RGB).