Protease inhibitor cocktail set 3

10⁵ SH-SY5Y or SNCA-EGFP gene-transfected SH-SY5Y cells were seeded into 6-well plates for 24 h. Then, the cells were incubated with each artificial enzyme sample (100 μg mL⁻¹ in 1 mL medium) for 36 h. Then, cells were lysed in cell lysis buffer for Western containing protease inhibitor cocktail set III (beyotime). The protein concentrations in lysates were quantified by BCA protein assay kit. 15 μg protein extracts were separated using 10% SDS-polyacrylamide gels. The proteins in the gel after electrophoresis were transferred onto PVDF membrane, and the nonspecific binding sites were blocked by 5% milk/TBST for 1.5 h. The membranes were incubated with the primary antibody for SNCA (Cell Signaling Technology, validated by manufacturer, cat# 51510, clone number E4U2F, 1:1000 dilution) at 4 °C overnight, washed several times, and then incubated with the HRP-labeled anti-rabbit secondary antibody (Cell Signaling Technology, validated by manufacturer, cat# 7074, 1:1000 dilution) for 1 h, and then subjected to ECL detection analysis.
To quantify SNCA in mouse brains, total tissue proteins were extracted from the cerebral hemispheres using the above-mentioned cell lysis buffer. The subsequent operation methods were performed as described above.

Thoracic aorta samples (0.5 g) were homogenized in 1 mL of RIPA lysis buffer (Beyotime Institute of Biotechnology, China) containing the protease inhibitor cocktail set III (Beyotime). Tissue lysates were incubated on ice for 30 min before the lysates were centrifuged at 12,000 ×g for 20 min at 4 °C. The supernatant was collected, and the protein concentrations were measured using a Coomassie Blue Staining Kit (Beyotime), with bovine serum albumin as the standard. Samples containing 50 μg of protein were resolved by 10% sodium dodecyl sulfate PAGE, electrotransferred onto a nitrocellulose membrane and incubated with antibodies against eNOS, t-PA, PAI-1 (Bioworld, USA) and GAPDH (Beyotime), according to the manufacturer’s instructions. Protein bands were developed with a horseradish peroxidase system (Beyotime). Protein bands were visualized by enhanced chemiluminescence with a BeyoECL Plus kit (Beyotime). The resulting images were resolved with Kodak X-Omat BT film (5 × 7IN; Beyotime). Band intensity was quantified by Gel-Pro Analyzer software (Liu Yi, Beijing). GAPDH was used as the loading control.