Whatman filter

The nitrocellulose membrane and Whatman filters (all GE Healthcare, Chicago, Il, USA) were washed 2X with Milli-Q H20 and 2X with Tris-Buffered Saline (TBS) by vacuum filtration. 450uL of each fraction sample was spotted on nitrocellulose membrane and washed 2X with TBS by vacuum filtration, and then allowed to air-dry. The blots were stained with Ponceau S solution and blocked with 5% nonfat milk in PBS/0.05% Tween 20 for 1 h at RT and subsequently incubated at 4°C overnight with the following primary antibodies: anti-ataxin-3 (1:2000; ProteinTech, Rosemont, IL, USA) and LaminB (1:2000; ProteinTech, Rosemont, IL, USA). The blots were probed with the respective HRP-conjugated secondary antibody (anti-rabbit or anti-mouse, 1:2000; Jackson Immuno Research, Suffolk, UK). The immunoreaction was detected using the ECL substrate (Thermo Fisher Scientific, Waltham, MA, USA). Data was collected using ChemiDoc XRS+ System with Image Lab v5.2 Software (Bio-Rad). To avoid overexposure of any band or dots, image acquisition times were set based on image histograms. Images were not processed before quantitation. Data within a membrane were normalized to total protein (Ki21 vs. Ki150).

Size-exclusion chromatography was performed in running buffer [veronal-buffered (VB) saline with added 140 mM NaCl]. MBL-deficient serum was reconstituted with rMBL [final concentration 50 µg/ml and incubated at room temperature (RT) for 60 min and, like MBL-sufficient and MBL-deficient sera, double filtered with a 0.2 µM Whatman filter (GE Healthcare, Buckinghamshire, UK)], before 500 µl sample was applied to 23.5 ml, 10 mm × 300 mm Superdex™ 200 10/300 GL (GE Healthcare, Buckinghamshire, UK) prepacked column equilibrated and eluted with running buffer at a constant 0.5 ml/min flow rate. 500 µl fractions were collected in 96-deep well plates and stored at 4°C until tested. The column was calibrated with GE Filtration Calibration Kits (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s guidelines.

Y. lipolytica was pregrown overnight in either YNB or YNB-PepYe. Cultures were passaged into fresh medium to an OD₆₀₀ of 0.05 and grown for 5 days at 28°C and 200 rpm. Total lipids were extracted using a modified form of the protocol originally developed by Bligh and Dyer (69 (link)). From each sample of Y. lipolytica culture, 10 ml was collected by vacuum filtration on a 0.45-μm nylon Whatman filter (GE Healthcare, Little Chalfont, United Kingdom) and washed thrice with 5 ml of yeast nitrogen base (1.7 g/liter) without amino acids and ammonium sulfate. The cells were mixed with 1.2 ml H₂O, 3 ml methanol, and 1.5 ml chloroform in a glass vial and mixed on a rotator for 24 h. Then 1.2 ml H₂O and 1.2 ml chloroform were added to the sample and briefly vortexed prior to separation of the organic and aqueous phases by centrifugation at 1,400 × g for 10 min. The organic phase was collected into a preweighed glass vial, and the extraction step was repeated three more times. The lipid-containing organic phase was dried at 40°C under a continuous stream of nitrogen gas and then weighed. Ten milliliters of culture from the same flask was separately collected and dried at 70°C to determine dry weight.

To obtain OMVs from B. bronchiseptica, bacteria were grown overnight to an OD₅₉₀ of approximately 1.5. Before OMV isolation was initiated, bacteria were incubated for one hour either at 56°C or with 0.5 μM of PMAP-36 at 37°C. Subsequently, bacterial cells were removed by centrifugation for 30 min at 4700 x g. The supernatant was passed through a 0.45 μm Whatman filter (GE Healthcare, Chicago, Illinois, USA) and centrifuged at 40,000 rpm for 2 h at 4°C (Ti-70 rotor, Beckman coulter, Brea, California, USA). The supernatant was decanted, and the transparent pellet was dissolved in 2 mM Tris-HCl (pH 7.5, Sigma-Aldrich) in a volume corresponding to 2% of the bacterial culture.