Western Blot Protein Quantification

As previously described [26 (link)–28 (link)], total cells and tissues were lysed using RIPA lysis 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 blocked with 5% nonfat milk in TBS for 3 h and incubated with various primary antibodies overnight at 4°C. After incubation with HRP-conjugated secondary antibodies (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 ChemiDoc MP imaging analysis system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The specific protein expression levels were normalized to β-tubulin on the same nitrocellulose membrane. The following primary antibodies and dilutions were used: anti-renalase (GTX89570, diluted 1 : 1000) was purchased from GeneTex (Irvine, CA, USA); anti-β-tubulin (sc-9104, diluted 1 : 2000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).