Sepharose 4b beads

2 µg purified αSN monomer or αSN oligomer was incubated with 1 mg/ml synaptosomal lysate, synaptosomal membranes, synaptic cytosol, or synaptic vesicles in PBS, 0.5% Triton X-100 in a total volume of 1 ml at 4°C in rotating tubes overnight. Affinity purified rabbit poly-clonal αSN specific antibody (ASY-1) covalently bound to Sepharose beads 4B (GE Healthcare) was added in a 5 times molar excess (5 IgG: 1 αSN) to the samples followed by 2 hours incubation at 4°C in rotating tubes. The Sepharose beads were isolated and washed twice with PBS, 0.5% Triton X-100, and Co-IP proteins were eluted by incubation in non-reducing SDS loading buffer for 2 hours at room temperature. Three individual Co-IP were conducted in the four described synaptosomal preparations using either monomeric, oligomeric αSN, or buffer as negative control, resulting in 36 samples to be analyzed by mass spectrometry.

Young (3 months) and old (22 months) heterozygous SNCA mice were sacrificed by the cervical dislocation and brain tissues were homogenized in ice-cold lysis buffer (T-PER^® tissue protein extraction reagent, ThermoFisher Scientific, USA) containing 1X proteinase inhibitor cocktail. Homogenized tissues were incubated in ice for 10 min and centrifuged at 10,000 g for 5 min at 4°C. The protein concentration of brain lysate was determined by Bicinchoninic acid (BCA) protein assay (ThermoFisher Scientific, USA). 3 mg of brain lysates were immunoprecipitated with Sepharose beads 4B (GE Healthcare, UK) pre-incubated with anti-VAMP2 antibody (Abcam, USA) at 4°C overnight, then immunoblotted using anti-α-syn antibody (BioLegend, 1:1000).

Human isoform of amphiphysin 2/BIN1 including exon 11 was expressed in Rosetta 2 bacteria and purified by affinity chromatography using glutathione Sepharose 4B beads according to the manufacturer’s instructions (GE Healthcare) in 50 mM Tris at pH 8.0, 100 mM NaCl. Proteins were expressed overnight at 18 °C using 1 mM IPTG induction, dialyzed overnight in a Slide-A-Lyzer dialysis cassette (MWCO 10,000) before Alexa Fluor 488 or 647 maleimide labeling (Invitrogen). Protein concentrations were measured using Bradford assay kits (Biorad).

Immunoprecipitated CERKL-Flag or GFP-Flag were incubated overnight at 4°C with m⁷-GTP-Sepharose or Sepharose-4B beads (GE Healthcare Life Sciences) in the immunoprecipitation lysis buffer. Sepharose beads were centrifuged and washed three times in the same buffer containing 450 mM NaCl. Proteins were eluted by incubating the beads in SDS-PAGE loading buffer at 70°C for 20 min, separated from the beads using the Pierce Spin Cups with cellulose acetate filter (Thermo Fisher Scientific) and then analyzed by Western blot.

CHO-sCR3 cells were cultured in F12 medium supplemented with 10% FCS at 37°C and culture supernatants were harvested every week. sCR3 was purified from the filtered culture supernatant by using an immunoaffinity chromatography matrix prepared by a covalent linkage of MEM-174 mAb to Cyanogen bromide (CNBr)-activated Sepharose 4B beads (GE Healthcare, Piscataway, NJ). The bent and extended conformers of the sCR3 ectodomain were separated by gel filtration chromatography on Superdex 200 HR (GE Healthcare, Piscataway, NJ) in HBSS buffer complemented with 2 mM CaCl₂ and 2 mM MgCl₂. The freshly separated conformers of sCR3 were directly applied on glow-discharged carbon copper grids (Benada and Pokorny, 1990 (link)), stained with 0.75% uranyl formate, and visualized in a Philips CM 100 transmission electron microscope (FEI, Eidhoven, Netherlands) equipped with a MegaViewII slow scan camera controlled by AnalySis 3.2 software (Olympus Soft Imaging Solutions, Münster, Germany).

The GST fusion proteins (GST-Hax-1-D1-D5) and the respective GST-fusion protein bound glutathione Sepharose 4B beads (GE Health Care) were prepared following previously published methods [48 (link)]. For the pull down assay, SKOV3 cells were lysed in a GST-lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 8.0), 1mM MgCl₂, 0.1% NP40, 10% glycerol incubated with the GST fusion protein domains D1-D5 of Hax-1 for 4 hours at 4°C, washed five times, and eluted in SDS sample loading buffer for analysis by western blotting.