α syn

The proteins of brain tissues or MN9D cells were extracted using RIPA buffer (ThermoFisher Scientific, CA, USA). BCA assay (Beyotime Biotechnology, Nantong, China) was used to quantify protein concentrations. After boiling for 5 min at 95°C, the protein samples (50 μg) were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF; ThermoFisher Scientific, CA, USA). The membranes were blocked with 5% skim milk for 1 h at room temperature. Then, the membranes were incubated with the primary antibodies against α-syn (ThermoFisher Scientific, CA, USA), SP-1 (ThermoFisher Scientific, CA, USA) or β-actin (Abcam, MA, USA) at 4°C overnight. The membranes were then incubated with the horseradish peroxidase-conjugated secondary antibodies (Abcam, MA, USA) for 2 h at room temperature. The blotted proteins were visualized using the ECL Western blotting substrate (ThermoFisher Scientific, CA, USA) and the intensity of band was quantified using image software (Bio-Rad, CA, USA).