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The protein concentration was verified by the method of Lowry33. Cells and kidney tissues were lysed with a 200-µL RIPA lysis buffer per plate (100 mm²). The lysates were centrifuged at 12,000 g for 5 min at 4ºC, and the supernatants were stored at −80ºC. Proteins (30 µg) were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes using a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad, CA, USA). Nonspecific binding sites were blocked with 5% albumin (v/v) in TBS buffer. The immunoblots were incubated overnight at 4ºC with renin (1:500, Santa Cruz, TX, USA), angiotensin I (1:500, Santa Cruz, TX, USA), angiotensin II (1:500, Santa Cruz, TX, USA), or GAPDH (1:500, Abcam, MA, USA) primary antibodies. After washing three times with TBS-T, the membranes were incubated for 1 h at 4ºC in HRP-conjugated secondary antibodies (1:100,000; Santa Cruz TX, USA). Immunoreactive protein bands were visualized using Pierce ECL Plus Chemiluminescent substrate (Thermo Fisher, USA). Images were obtained and analyzed with an Alliance 7 Chemiluminescence documentation system (UVITEC, Cambridge, UK). The immunoblot band intensities were quantified using Image J software and reported as the renin/GAPDH, Angiotensin I/GAPDH, and Angiotensin II/GAPDH ratio.