Figure 2

HNE-induced aggregation of WT and H50 mutant aSyn. A) Recombinant human WT aSyn was treated with 0, 0.2, 1, and 3 mM HNE for 24 h and analyzed by SDS-PAGE followed by WB using an antibody against aSyn (Syn-1). The formation of SDS-resistant aSyn oligomers as indicated shows an increase in a HNE-concentration dependent manner. M: aSyn monomers; D: aSyn dimers; O: aSyn oligomers. B) Recombinant human WT and H50Q/R aSyn were treated with 3 mM HNE for 24 h and analyzed by SDS-PAGE and WB. H50Q/R mutations decrease the formation of HNE-induced SDS-resistant aSyn oligomers as probed by two different antibodies raised against distinct aSyn antigenes (Syn-1 antibody, left, and SNCA antibody, right). C) WT, H50Q, and H50R aSyn were treated with 3 mM HNE for 24 h and subsequently analyzed by SEC in order to assess the formation of soluble oligomers. aSyn monomers (M) and oligomers (O) are distinguished by different retention times. D) Area ratio of oligomeric to monomeric fraction in HPLC chromatograms of SEC was calculated for aSyn incubated with 0.2, 1, or 3 mM HNE. H50 mutations reduce the formation of HNE-induced soluble oligomers. Significance was determined by a one way ANOVA Tukey’s Multiple Comparison Test. n = 3. Error bars = SEM.