Protein Extraction and Western Blot Analysis

We used the previously described method to extract proteins from the worms and perform Western blot analysis [31 (link)]. Protein was extracted from frozen worm pellets using Fastprep24 (MP Biomedicals LLC, Solon, OH, USA) and PBS containing protease inhibitors. The extract was boiled with sample buffer containing sodium dodecyl sulfate (SDS) for 10 min and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. After reacting with the primary antibody overnight, the position and intensity of human α-synuclein were determined by use of horseradish peroxidase (HRP)-conjugated secondary antibody (PerkinElmer Inc., Boston, MA, USA) and the Amersham enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA) and BioSpectrum imaging system (UVP, Upland, CA, United States). Human α-synuclein monoclonal antibody (sc-12767) and β-actin (sc-47778) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).