Western blot on 15ug mouse brain lysate from Fmr1 KO mice (FXS mice) with C57Bl/6J genetic background. Block: 5% Skim Milk in 1X TBST. Primary Antibody: Mouse Anti-Alpha Synuclein Monoclonal Antibody clone 2F11 at 1:1000 for 2 hours at RT. Secondary Antibody: Goat Anti-Mouse HRP:IgG at 1:3000 for 1 hour at RT. Color Development: ECL solution (Super Signal West Pico) for 5 min in RT. Band clearly identifies large molecular weight oligomeric band (monomeric band predicted at 14kD).

Western blot on 15ug Hela lysate. Block: 5% Skim Milk in 1X TBST. Primary Antibody: Mouse Anti-Alpha Synuclein Monoclonal Antibody clone 2F11 at 1:1000 for 2 hours at RT. Secondary Antibody: Goat Anti-Mouse HRP:IgG at 1:3000 for 1 hour at RT. Color Development: ECL solution (Super Signal West Pico) for 5 min in RT. Band clearly identifies large molecular weight oligomeric band (monomeric band predicted at 14kD).

SPR binding analysis of 2F11 antibody and human alpha synuclein type 1 fibrils (SPR-322), type 2 (SPR-317) fibrils and monomeric protein (SPR-321). Buffer (top left), alpha synuclein monomers (top right), alpha synuclein fibrils type1 (bottom left), and alpha synuclein fibrils type 2 (bottom right). Data: KD[nM] for SPR-322 = 42.5, SPR-317 = 365.3; 2F11 binds SPR-322 with 8.6 x greater affinity than SPR-317. 2F11 does not bind monomeric alpha synuclein (SPR-321).

8 Day differentiated iPSCs with added alpha synuclein fibrils (SPR-322), conjugated to Alexa Fluor 555 with or without 1h prior 2F11 addition. SPR-322 Alexa Fluor 555 added at 5µg/well for 48h, 2F11 (50ug/well added 1h before fibrils where applicable). Cells were subsequently washed 48h after addition and imaged a following 24h later. Imaging and channels: 2F11 antibody: 647nm (using an anti-mouse-647 conjugate); Fibrils: 555nm, phosph-ser129 (SPR-600) 488nm, and Hoechst stain for DNA. Image 4a top, no fibrils, no 2F11 added. Bottom, fibrils added, no 2F11 antibody. Fibrils are clearly visible in the cells as is enhanced phosph-ser129 signal. Image 4b top, no fibrils, 2F11 antibody added. Bottom, fibrils and 2F11 antibody added. The image clearly shows 2F11 antibody addition stops cell entry of the 555-labelled fibrils.