Western Blot Analysis of Renalase

As described in previous studies [15 (link), 16 (link)], total tissues were lysed using RIPA buffer, and the protein concentration was determined with a BCA protein assay kit (Pierce Company, Rockford, IL, USA). Protein extracts were used for SDS-PAGE (Invitrogen, Carlsbad, CA, USA), and the proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), which was incubated with primary antibodies (renalase GTX89570, GeneTex, diluted 1 : 1000) overnight at 4°C. After incubation with secondary antibodies (goat IgG antibody GTX228416-01, GeneTex, diluted 1 : 5000) for 1 h at room temperature, the membranes were treated with ECL reagents (170-5061, Bio-Rad, Hercules, CA, USA) prior to visualization using a FluorChem E imager (Cell Biosciences, San Jose, CA) according to the manufacturer's instructions. The specific protein expression levels were normalized to β-tubulin on the same nitrocellulose membrane.