Amersham enhanced chemiluminescence system

The SDS-PAGE-separated proteins were transferred onto 0.45 μm pore-size PVDF membranes (Millipore; Burlington, MA, USA) using Trans-Blot (Bio-Rad; Hercules, CA, USA). The membranes were blocked in 5% zero-fat-dried milk in Tris-buffered saline containing 0.1% Tween 20 prior to the incubation in primary antibodies for 1–3 h at room temperature or overnight at 4 °C. The membranes were subjected to several washes in Tris-buffered saline containing 0.1% Tween 20 (TBST) to remove excess antibodies. The immunoreactive proteins were detected by incubation with a species-specific horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The immune complexes were detected with the Amersham Enhanced ChemiLuminescence system (Amersham Biosciences; Piscataway, NJ, USA) prior to scanning using the Fluor-S Max MultiImager system (Bio-Rad; Hercules, CA, USA).

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).

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).