Fig 1: Characterization of binding properties between a-synuclein PFFs and LAG3 reveals promiscuous character of a-synuclein A, BBinding of ASC filaments (A) or a-synuclein PFFs (B) to multiple proteins. Serial dilutions of ASC filaments and a-synuclein PFFs were performed, and bound fractions were detected using anti-ASC or anti-a-synuclein (MJFR1) antibodies. ASC filaments did not interact with any of the proteins (A). Conversely, a-synuclein PFFs strongly bound LAG3, CD4 and MTBD tau and showed moderate binding properties to PrPC, APOE3 and TDP-43. No binding between a-synuclein PFFs and nAra h 2 or BSA could be detected. Values represent means ± SD of technical duplicates.C, DDiffusional sizing of a-synuclein PFFs with fluorescently conjugated LAG3, CD4, BSA and MJFR1 a-synuclein antibody (C) and of fluorescently labelled LAG3 with monomeric a-synuclein, a-synuclein PFFs, FGL1 and Relatlimab anti-LAG3 antibody (D). An increase in the hydrodynamic radius (Rh) of a-synuclein PFFs is seen with increasing concentrations for MJFR1 but not for LAG3 or any of the control proteins, also not at a lower scale (see insert) (C). Similarly, the hydrodynamic radius of labelled LAG3 increases with higher concentrations of Relatlimab and FGL1 but does not change with a-synuclein PFFs or monomeric a-synuclein (D). Values are given as means ± SD of technical triplicates.E, FCo-immunoprecipitation of LAG3 and subsequent immunoblotting with anti-a-synuclein antibody (MJFR1) (E) and co-immunoprecipitation of a-synuclein (monomeric or PFFs) and subsequent immunoblotting with anti-LAG3 antibody (D2G40) (F). Using the anti-a-synuclein antibody for pulldown resulted in LAG3 bands with both monomeric as well as fibrillar a-synuclein. The usage of anti-LAG3 for pulldown led to the specific detection of a-synuclein PFFs, although the PFF fraction predominates also in the input and supernatant. Bands for a-synuclein (E) and LAG3 (F) are indicated by arrows. Source data are available online for this figure.