Bradford reagent

C. neoformans H99 (serotype A) and 1841 (serotype D) cells were recovered from 10% skim milk stocks stored at -80°C and grown independently for 48 h in Sabouraud dextrose broth while shaking (80 rpm) at 30°C. Cells were harvested by centrifugation and washed twice with 250 mM sucrose. The pellet was resuspended in lysis buffer [5 mM Tris/HCl pH 7.5, 2.5 mM EDTA, 0.5X protease inhibitor cocktail (Roche, Basel, Switzerland)] additionally containing 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, Cat. No. 1479, Carl Roth, Karlsruhe, Germany) and 50 mM dithiothreitol (DTT, Cat. No. 6908, Carl Roth, Germany). The cell suspension was transferred into a mortar, frozen with liquid nitrogen and homogenized with a pestle twice. Afterwards, homogenates were centrifuged and supernatant was recovered. Protein content was estimated using Bradford reagent (Carl Roth, Karlsruhe, Germany). Proteins were precipitated with 10% trichloroacetic acid over night at -20°C and centrifuged. After removal of the supernatant, the pellet was washed three times with ice-cold acetone and air-dried. The protein pellet was dissolved in a solution containing 7 M urea, 2 M thiourea, and 4% CHAPS and protein content was estimated using Bradford reagent (Carl Roth, Germany).

Subcellular protein fractions were isolated using a ready-made kit following the instructions by the manufacturer (ThermoFisher Scientific). Granulocyte proteins and proteins of enzymatically treated isolated nuclei were isolated after adding HALT protease inhibitor (ThermoFisher Scientific) directly to the culture well and subsequent prompt heat denaturation at 95°C for 5 min in LDS gel loading buffer. Protein quantification was performed using Bradford reagent (Carl Roth). For further analysis, Western blots were performed after SDS-PAGE using ready-made gels (Bio-Rad). Digital image acquisition was performed using a Bio-Rad ChemiDoc Imaging System (Bio-Rad). Images of molecular size markers and specific Western Blots were merged using the manufacturer's recommended software. Nitrocellulose membranes of western blots were reprobed after washing in Tris buffered saline, 0.1% Tween-20 (TBS/T) and stripping in Tris-HCl pH 6.8, SDS and β-mercaptoethanol.

Mice were 6 months old when sacrificed by cervical dislocation (2 wild-type and 3 L62 mice, Table 1; Experiment I-B). The top of the skull was exposed, and the overlying bone plates removed to allow harvest of the whole brain. The cortex and the striatum were dissected, transferred into separate 1.5 mL reaction tubes, immediately frozen in liquid nitrogen, and kept at − 80 °C until use. Additionally, spinal cord tissue was dissected, snap frozen and stored at − 80 °C. Tissue from each mouse was crushed and used for urea extraction according to protocol 1 as follows. Crushed brain tissue was incubated for 45 min at RT in six volumes of urea extraction buffer (7 M urea, 2 M thiourea, 2% ampholyte 2–4, 70 mM DTT, 25 mM Tris/HCl pH 8.0, 50 mM KCl, 3 mM EDTA, 2.9 mM benzamidine and 2.1 µM leupeptin) and separated by centrifugation at 16,000×g for 45 min at RT. The supernatant was transferred to new tubes and used for electrophoresis. The protein concentration in the supernatants was determined using the Bradford Reagent (Carl Roth, Karlsruhe, Germany) according to the manufacturer´s recommendations. The urea supernatants were used for antibody specificity studies (Experiment I-B), and blots were labelled using the monoclonal anti α-Syn antibodies syn204, mAbs 559, 874 and 736, Clone 42, mAb 211, 3H2897, C20 and Ab1903 (see Table 2 for details).