Fig. 2
Alpha-synuclein activates the classical complement cascade. A Absorbance profile at 214 nm of purified α-synuclein (α-syn) subjected to size-exclusion chromatography analysis. Monomeric α-syn elutes as a 60-kDa protein due to its natively unfolded state. B Wells coated with α-syn were incubated with normal human serum (NHS) or NHS in the presence of EDTA (Ca2+ depleted). The deposition of complement was measured using a biotinylated C4b-fragment-specific antibody followed by incubation with streptavidin-europium. Time-resolved fluorescence from the europium is given as million counts per second (mcps). Statistical test: area under the curve (AUC) was compared by a ratio paired t-test. C Same procedure as B except wells were coated with either human serum albumin (HSA), α-syn, or α-syn depleted for lipopolysaccharide (LPS depleted) followed by incubation with NHS. Statistical test: AUC was compared by a one-way ANOVA for repeated measures followed by Tukey’s multiple comparison. D Same as B, except wells were coated with HSA, α-syn, Tau, or carbonic anhydrase followed by incubation with NHS. Statistical test: AUC was compared by a one-way ANOVA for repeated measures followed by Tukey’s multiple comparison. Aα-Syn compared to Tau, carbonic anhydrase, and HSA. E Same as B, except wells were coated with HSA, α-syn, β-synuclein (β-syn), or C-terminal truncated α-syn (α-syn(1-95)) followed by incubation with NHS. Statistical test: AUC was compared by a one-way ANOVA for repeated measures followed by Tukey’s multiple comparison. Bα-Syn and β-syn compared to HSA. F Same as B, except α-syn-coated wells were incubated with C1q-depleted NHS (C1q depleted) without or with added C1q. Statistical test: AUC was compared by a ratio paired t-test. All data points are presented as mean ± SD. B, C, E, F n = 3; D n = 5