Mouse anti αsyn antibody

48 h after transfection, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). After washing with PBS, cells were permeabilized with 0.5% Triton X-100/PBS (Sigma-Aldrich) for 20 min at RT, and blocked in 1.5% normal goat serum (PAA)/PBS for 1 h. Cells were incubated with a mouse anti αSyn antibody (1:1000, BD Transduction Laboratory) overnight and then with a secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG) for 2 h at RT. Finally, cells were stained with Hoechst 33258 (1:5000 in PBS, Life Technologies- Invitrogen) for 5 min and maintained in PBS prior to epifluorescence microscopy.

Blotted aliquots of αSyn (10 × 2 μl) were dried on nitrocellulose before being blocked for 1 h in Tris‐buffered saline (TBS: 10 mm Tris; 150 mm NaCl; pH 7.5) + Tween20 (TBST) containing 2% dried skimmed milk (Marvel; Premier Foods, St Albans, UK). Primary antibodies were diluted 1:1000 and secondary antibodies were diluted 1:5000 in the blocking buffer. Mouse anti‐αSyn antibody (catalogue number 610786; BD Biosciences, Clontech, Palo Alto, CA, USA) was used to detect the presence of αSyn regardless of structure, the secondary for which was anti‐mouse IgG conjugated to alkaline phosphatase (Apase; Promega, Madison, WI, USA). Rabbit polyclonal A11 antibody (catalogue number AHB0052; Invitrogen, Carlsbad, CA, USA) was used to specifically detect oligomeric αSyn (Kayed et al. 2003), the secondary for which was anti‐rabbit IgG conjugated to APase (Promega). Before and after the addition of secondary antibodies, the paper was washed thoroughly in TBST to remove non‐specific binding. A final wash in TBS was carried out to remove Tween20 prior to the addition of Western Blue (Promega), a substrate for APase, that enabled visualization of the target protein.