Antibodies used for western blotting were as follows: anti-Flag M2 (1:10000, Sigma-Aldrich, St. Louis, MO, USA); anti-NSF (1:500, Cell Signaling, Danvers, MA, USA); anti-LRRK2 (1:1000, C41-2, Abcam, Cambridge, UK); anti-Synaptobrevin, anti-synaptophysin and anti-Synaptotagmin 1 (1:1000, Synaptic System, Göttingen, Germany).
Between 10 and 20 μg of protein samples were dissolved in 4–20 % Tris-glycine polyacrylamide gels (Biorad) in SDS/Tris-glycine running buffer. Precision Plus molecular weight markers (Biorad) were used for size estimation. Solubilized proteins were then transferred to polyvinylidenedifluoride (PVDF) membranes in transfer buffer containing 10 % methanol. The PVDF sheets were blocked in Tris-buffered saline plus 0.1 % Triton (TBS-T) plus 5 % nonfat dry milk for 1 h at 4 °C and then incubated overnight at 4 °C with primary antibody in TBS-T plus 5 % non-fat dry milk. The PVDF membranes were washed in TBS-T (3 × 10 min) at room temperature (RT) followed by incubation for 1 h at RT with horseradish peroxidase-conjugated anti-mouse IgG. Blots were then washed in TBS-T (4 × 10 min) at RT and rinsed in TBS, and immunoreactive proteins were visualized using enhanced chemiluminescence plus (ECL+, GE Healthcare, Waukesha, WI, USA). Densitometric analysis was carried out using Image J software.
Between 10 and 20 μg of protein samples were dissolved in 4–20 % Tris-glycine polyacrylamide gels (Biorad) in SDS/Tris-glycine running buffer. Precision Plus molecular weight markers (Biorad) were used for size estimation. Solubilized proteins were then transferred to polyvinylidenedifluoride (PVDF) membranes in transfer buffer containing 10 % methanol. The PVDF sheets were blocked in Tris-buffered saline plus 0.1 % Triton (TBS-T) plus 5 % nonfat dry milk for 1 h at 4 °C and then incubated overnight at 4 °C with primary antibody in TBS-T plus 5 % non-fat dry milk. The PVDF membranes were washed in TBS-T (3 × 10 min) at room temperature (RT) followed by incubation for 1 h at RT with horseradish peroxidase-conjugated anti-mouse IgG. Blots were then washed in TBS-T (4 × 10 min) at RT and rinsed in TBS, and immunoreactive proteins were visualized using enhanced chemiluminescence plus (ECL+, GE Healthcare, Waukesha, WI, USA). Densitometric analysis was carried out using Image J software.