Bis tris gel

Proteins were separated by electrophoresis using 10% or 4–12% Bis-Tris gels (GenScript) and subsequently stained using Pierce Silver Stain Kit (Thermo Fisher) or used for Western blotting. Protein transfer to nitrocellulose membranes was done using standard procedures. Membranes were blocked with TBST buffer (Tris-buffered saline, pH 7.4, with 0.05% Tween-20 containing 5% (w/v) non-fat dry milk) for 1 h at room temperature; primary antibody was incubated overnight at 4 °C and secondary antibody for 1 h at room temperature. Membranes were developed with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher) and visualized by autoradiography.

α-Syn fibrils were concentrated by centrifugation (14,462× g, 25 °C, 45 min), washed and resuspended with PBS, followed by sonication at 20% power for 15 times (1 s per time, 1 s interval) on ice by JY92-IIN sonicator. α-Syn PFFs (3 mg ml⁻¹, 25 μl, in PBS, PH 7.4) were incubated with proteinase K (final concentration 0.5, 1, 1.5, 2, 2.5 μg ml⁻¹, Invitrogen) at 37 °C for 30 min. Reaction was terminated by adding 1 mM PMSF. The samples were boiled with SDS-loading buffer for 15 min and loaded on 4–20% Bis-Tris gels (GenScript). The gels were stained by Coomassie brilliant blue and images were recorded and analyzed with Image Lab 3.0 (Bio-Rad).

SDS-PAGE was performed with 4–20 % bis-Tris gels (Genscript) followed by protein staining/destaining. sEC-HPLC was performed using a Waters Alliance system and a Sepax sEC-150 column (3μ particle size, 7.8 × 300), equilibrated with PBS + 0.02 % sodium azide and run at 1 mL/min. Detection was at 280 nm. Primary amine concentrations were determined using trinitrobenzene sulfonic acid (TNBS) [29 (link)]. Lf assay was performed at the National Institute for Biological Standards and Control (United Kingdom), using the 3rd International Standard tetanus toxoid for use in flocculation test as the reference standard and 66/021 Equine tetanus antitoxin as the flocculation antibody.

The cells were washed using an ice-cold phosphate-buffered saline (PBS) and then lysed with the Membrane and Cytosol Protein Extraction Kit (20127ES60 Yeasen, China). The kit was further supplemented with protease (20124ES03 Yeasen, China) and phosphatase inhibitors (20109ES05 Yeasen, China). The concentration of the proteins was determined via the BCA Protein Assay Kit (20201ES76 Yeasen, China), and the process was conducted in accordance with the manufacturer’s instructions. The proteins were segregated on 4–20% Bis-Tris gels (Genscript, China), subsequently being transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore ISEQ00010, China). The membranes were then blocked using 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 1 hour. Primary antibodies were diluted in 0.5% non-fat milk in TBST and were then allowed to incubate with the membrane at 4°C overnight: Anti-LTBR antibody (Absin abs146148, 1:1,000), Anti-GAPDH (Absin abs830030, 1:2,000). After three washes with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (34201ES60 Yeasen, China) in TBST at room temperature for 1 hour. The immunoreactive bands were then visualized utilizing the Enhanced Chemiluminescence (ECL) Western Blotting Substrate (36208ES60 Yeasen, China).