Quantification of CX43 and VEGFA Protein Levels

The renal cortexices from the diabetic and non-diabetic kidneys were lysed with protease and phosphatase inhibitors to prevent protein degradation and stored at −80 °C until analysis. Fifty microgram of total protein lysates were denatured, resolved using 10% SDS-PAGE, and transferred to a nitrocellulose membrane, as described in detail previously [18 (link)]. The membrane was blocked with 5% dry milk and incubated overnight with rabbit anti-CX43 antibody at 1:6000 (#C6219, Sigma), or anti-VEGFA at 1:200 (#sc-7269; Santa Cruz Biotechnology, TX, USA). After incubation with a horseradish peroxidase–conjugated secondary IgG (Sigma), the blots were developed using the SuperSignal™ West Femto Maximum Sensitivity Substrate (#34095; Thermo Scientific, MI, USA). Chemiluminescent signals were captured using an ImageQuant LAS 4000 Imager (GE Healthcare Bio-Sciences AB, Sweden) and analyzed by ImageJ software (http://imagej.nih.gov/ij/download.html). Ponceau S staining was used as the loading control.