Pierce ecl plus chemiluminescent substrate detecting reagents

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 4 °C, and the supernatants were stored at −80 °C. 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). Nonspecific binding sites were blocked with 5% albumin (v/v) in a TBS buffer. The immunoblots were incubated overnight at 4 °C with the TGF-β1 or GAPDH primary antibodies (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). After washing three times with TBS-T, the membranes were incubated for 1 h at 4 °C in HRP-conjugated secondary antibodies (1:30000; Cell Signalling). Immunoreactive protein bands were visualized using Pierce ECL Plus Chemiluminescent substrate detecting reagents (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 expressed as the TGF-β1/GAPDH ratio.

The protein concentration was verified by the method of Lowry [66 (link)]. IMCD cells 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 4°C, and the supernatants were stored at −80°C. Proteins (30 μg) were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes using a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad). Nonspecific binding sites were blocked with 5% BSA (v/v) in a TBS buffer. The immunoblots were incubated overnight at 4°C with the TGF-β1 and GAPDH primary antibodies (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). After washing three times with TBS-T, the membranes were incubated for 1 h at 4°C in HRP-conjugated secondary antibodies (1:30000; Cell Signalling). Immunoreactive protein bands were visualized using Pierce ECL Plus Chemiluminescent substrate detecting reagents (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 ImageJ software and expressed as the TGF-β1/GAPDH ratio.