Anti ps129

Neurons were fixed with 4% paraformaldehyde (PFA) and 0.1% TX‐100 in PBS for 15 min. To detect the insoluble α‐syn aggregates, α‐syn‐HEK293 cells were fixed with 4% PFA in PBS followed by permeabilization with 1% TX‐100. Cells were incubated with anti‐pS129 (1:1000, Biolegend, 825701), anti‐Ubiquitin (1:500, Cell Signaling Technology, #3936) overnight at 4°C. The samples were stained with corresponding secondary antibodies Alexa Fluor 594 or 488 (1:1000, Invitrogen). Nuclei were visualized with DAPI (1 μg/ml, BioFroxx, 1155MG010) for 5 min. To quantify the percentage of positive cells, a total of 8 fields, each with 100+ cells, were analyzed per condition. The primary cultured neurons and brain sections were double stained for MAP2/pS129, pS129/K80Hcy, Thioflavin S (ThS)/K80Hcy, TH/pS129, TH/K80Hcy, or GFP/TH.

Lovastatin and Thioflavin T were purchased from Sigma (St. Louis, MO, United States). Lipofectamine-2000, β-D-thiogalactoside (IPTG), and Alexa Fluor 488/594-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Anti-p-S129 and anti-GAPDH were purchased from BioLegend (San Diego, CA, United States). Anti-CK2α, anti-CK2β, anti-Histone 3, and anti-Histone 4 were purchased from Proteintech (Wuhan, China). Anti-AcH3K9 and AcH4K5 were purchased from Cell Signaling Technology (Danvers, MA, United States). CK2 and GRK5 kits were purchased from Jianglaibio (Shanghai, China).