Western Blot Analysis of Podocyte Proteins

Cells were rinsed twice with PBS and lysed by M-PER Protein Extraction Buffer (Pierce, Rockford, IL) containing 1× protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Proteins (20–30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to an Immuno-Blot polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA). After blocking in PBS/Tween (0.1%) with 5% nonfat milk, the membrane was incubated with primary antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, 1∶3000) and then developed using Enhanced Chemiluminescent (ECL) solution (Pierce). Primary antibodies included rabbit anti-nephrin (Santa Cruz, 1∶1000), goat anti-synaptopodin (Santa Cruz, 1∶1000), mouse anti-CD2AP (Santa Cruz, 1∶1000), rabbit anti-podocin (Sigma, 1∶200), goat anti-actin (Santa Cruz, 1∶1000), rabbit anti-phospho-JNK (Cell Signaling, 1∶1000), rabbit anti-JNK (Cell Signaling, 1∶1000), rabbit anti-phospho-Akt (Cell Signaling, 1∶1000), rabbit anti-Akt (Cell Signaling, 1∶1000), rabbit anti-phospho-p38 (Cell Signaling, 1∶1000), and rabbit anti-p38 (Cell Signaling, 1∶1000). For data quantification, the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed using the public domain NIH image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/).