Protein Extraction and Western Blotting of α-Synuclein

We used Fastprep24 (MP Biomedicals LLC, Solon, OH, USA) with protease inhibitors/PBS to obtain protein extracts from frozen whole-worm pellets. Western blotting was performed by using a previously described protocol [76 (link)]. Samples were boiled 10 min with sodium dodecyl sulfate (SDS) sample buffer, and separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Antibody binding was visualized by binding of horse-radish peroxidase (HRP)-coupled secondary antibody and Amersham enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA). Signals were detected by using a BioSpectrum Imaging System (UVP, Upland, CA, USA) Human α-synuclein monoclonal antibody and β-actin antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).