Anti p α syn

Proteins were extracted by incubating brain tissues with co-immunoprecipitation lysis buffer (20 mM Tris (pH = 7.5), 150 mM NaCl, 1% Triton X-100), supplemented with freshly added protease inhibitor cocktail and phosphatase inhibitor cocktail, for 30 min on ice. The lysates were then centrifuged (13,700 × g) for 10 min at 4 °C. Subsequently, the supernatant was incubated overnight at 4 °C with rocking, along with anti-α-syn (cat. no. OM239190, Omnimabs), anti-VAMT2 (cat. no. ab259970, clone number: EPR24197-51, Abcam), anti-p-α-syn (cat. no. AF3285, Affinity Biosciences), or IgG (cat. no. 30000-0-AP, Proteintech). Then, samples were incubated with magnetic protein A/G beads (MedChemExpress) for 60 min at 4 °C with gentle rocking. The formed complexes were washed with chilled buffers, and then eluted by boiling in 5X loading buffer for western blotting analysis.

We first placed SH-SY5Y cells on coverslips with poly-D-lysine coating. Next, 4% paraformaldehyde was used to fix cells, followed by blocking with 5% normal donkey serum (Jackson ImmunoResearch, Bar Harbor, ME, USA) and 0.1% TX (Sigma-Aldrich) for 1 h at room temperature. To assess the expression levels of p-α-Syn, a series of incubations with the primary antibody anti-p-α-Syn (1:1000, Abcam) were conducted at 4 °C overnight. The samples were washed off with 0.1% TX in phosphate-buffered saline, followed by 1 h of incubation with a mixture of fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) and cyanine 3 (CY3)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) at room temperature. Fluorescent images were acquired using a confocal microscope (Zeiss Confocal LSM 710) after coverslips were mounted. All images were processed using Zeiss Zen software. The range of signal intensity of the threshold in the selected areas was measured using ImageJ software.