Lb509

One microliter of each sample, dot #1 (α-Syn monomer), dots #2–4 (α-Syn oligomeric polymorphs), dot #5 (α-Syn fibrils), dot #6 (tau oligomers), and dot #7/#8 (Aβ40/42 oligomers) was dotted on nitrocellulose membrane and let dry for 1 h at RT. Next, membranes were blocked with 10% nonfat milk in TBS-T overnight at 4 °C. After blocking, membranes were probed with primary antibodies SynTC1 (1:4000), SynTC2 (1:1000), SynTC3 (1:1000), and total α-Syn commercial antibody LB509 (1:5000; Abcam 27,766) in 5% nonfat milk for 1 h at RT followed by incubation with HRP-conjugated IgG anti-mouse (1:6000, GE Healthcare) secondary antibody to detect α-Syn antibodies. ECL plus (GE Healthcare) was used for signal detection [44 (link)].

Protein concentrations per brain sample were determined utilizing a Pierce BCA protein assay kit (Hu et al., 2022 ; Thermo Fisher). A total of 50 µg of protein were loaded into each well and resolved by gel electrophoresis. Separated proteins were transferred from the gel to a nitrocellulose membrane, which was verified via Ponceau S staining (Martin et al., 2006 (link)). Membranes were blocked in TBST (0.1% Tween 20 in TBS) with 5% BSA for 2 h and then incubated with alpha-synuclein monoclonal primary antibody (LB509; Abcam; 1:500) overnight at 4°C and then stained with a goat-anti-mouse secondary antibody (ab205719; Abcam; 1:20,000). After washing with TBST, membranes were incubated with secondary antibody for 2 h at room temperature. Membranes were washed again and then used for enhanced chemiluminescence utilizing the Bio-Rad Clarity Western ECL Substrate (Bio-Rad; Fig. 1E).

After heat denaturation (5 min at 100°C), the proteins were separated by electrophoresis using 12% SDS-polyacrylamide gels before being electroblotted onto polyvinylidene fluoride membranes (BioRad, Marnes La Coquette, France). αS was probed with either mouse monoclonal antibody (mAb) clone 42 against αS (BD Transduction Laboratories), LB509 against human αS (ref ab27766), rabbit mAb against pSer129 αS (ref ab51253; Abcam, Cambridge, UK) or rabbit polyclonal antibody (pAb) against pSer129 αS (ref ab59264; Abcam, Cambridge, UK) (Table 2). Anti-actin mAb (ref ab8226; Abcam, Cambridge, UK) and anti-βsyn (ref ab76111; Abcam, Cambrigde, UK) were used as controls. The membranes were then incubated with horseradish peroxidase conjugated goat anti-mouse (ref 32460; Thermo) or anti-rabbit (ref 32430; Thermo) Ig secondary antibodies (1:1000). The immunocomplexes were visualized with chemiluminescent reagents (Supersignal WestDura, ref 34076, Thermo), followed by exposure on Biomax MR Kodak films, or CL-Exposure films, and by analysis using the Versa Doc system and Quantity One software (both from BioRad).

ELISA plates were coated with 1 µg/well of α-Syn monomer, α-Syn oligomeric polymorphs, and tau oligomer. 0.1 M sodium bicarbonate, pH 9.6, was used as a coating buffer followed by overnight incubation with primary antibodies: SynTC1 (1:4000), SynTC2 (1:1000), SynTC3 (1:1000), and total α-Syn commercial antibody LB509 (1:5000; Abcam 27,766) at 4 °C. Plates were then washed three times with TBS-T and incubated with 100 μl of HRP-conjugated anti-mouse IgG, diluted in 5% nonfat milk in TBS-T, for 1 h at room temperature. Plates were washed three times with TBS-T and developed with 3,3,5,5-tetramethylbenzidine (S1599, Dako). The reaction was stopped using 100 μl of 1 m HCl, and absorbance was read at 450 nm using a POLARstar OMEGA plate reader. All experiments were performed in triplicate [42 (link), 43 (link)].

Following the 24-h incubation, cells were washed 3 times with 1 × PBS and fixed with 4% formaldehyde solution for 15 min at RT. Cells were then washed 3 times with 1 × PBS followed by permeabilizing with 0.25% Triton X-100 in PBS for 10 min at RT. Cells were blocked in 5% goat serum for 30 min at RT and incubated with primary antibodies: βIIITubulin (1:1000; Abcam78078), Syn10842 (1:1000; ThermFisher 10,842–1-AP), and total α-Syn antibody LB509 (1:5000; Abcam 27,766) at 4 °C overnight. The next day, cells were washed and incubated with Alexa-conjugated secondary antibodies (1:000; Life Technologies) for 1 h at RT in the dark. After three washes, cells were mounted with Prolong Gold antifade reagent with DAPI. Each treatment condition was performed in 3 replicates and were randomly imaged at five different regions of interest. Images were captured with a Keyence BZ-800 Microscope and analyzed using BZ-X Analyzer. A Nikon 60 × objective was used for image acquisition. To eliminate species cross-reactivity, fluorescent intensity of total α-Syn was quantified by total α-Syn anti-rabbit polyclonal, Syn10842 (1:1000; Thermo Fisher 10,842–1-AP). All images were analyzed by ImageJ (NIH) software. Statistical significance is measured by using two-way ANOVA with Bonferroni post hoc analysis. **p < 0.01, ****p < 0.0001. Scale bar 10 μm.

Three different concentrations of α-Syn oligomer preparations, as well fibrillar α-Syn sample were loaded on precast NuPAGE 4-12% Bis-Tris gels (Invitrogen) for SDS-PAGE analysis. For electrophoresis with tau aggregates, approximately 2 μg of each tau aggregate sample was loaded. Gels were subsequently transferred onto nitrocellulose membranes and blocked with 10% nonfat dry milk at 4 °C overnight. Membranes were then probed with primary antibodies, Syn33 (1:4000), LB509 (1:2000; Abcam, Ab27766), T22 (1:250), and Tau 5 (1:5000; BioLegend, 806402) diluted in 5% nonfat dry milk for 1 h at RT. HRP-conjugated, anti-mouse IgG (1:6000, GE Healthcare) was used to detect LB509 and Tau 5 immunoreactivity, whereas an HRP-conjugated anti-rabbit IgG (1:6000, GE Healthcare) was used for Syn33 and T22 immunoreactivity. ECL plus (GE Healthcare) was used to visualize the bands. Densitometric analysis was performed using ImageJ software (National Institutes of Health).