Fig 9. Effects of BrioHOCl on recombinant α-synuclein seeds and Lewy bodies.
Recombinant α-syn (rα-syn) fibrils generated in vitro (A) or Lewy Bodies isolated from a patient with Lewy Body Dementia (B) were treated with an active (190 ppm Cl) or inactive (30 ppm Cl) BrioHOCl solution at a 10:1 or 100:1 disinfectant to α-syn ratio for 5 or 60 min, as indicated, and probed for α-syn by immunoblot. Samples treated 10:1 were diluted an additional 10 fold in 1x sample buffer prior to loading the gel to match the protein concentrations of the 100:1 treated samples on the immunoblot. The arrow indicates monomeric α-syn protein and the bracket denotes aggregates and degradation products (A & B). Recombinant α-syn fibrils generated in vitro were treated with either a mock solution or an active BrioHOCl solution at 100:1 disinfectant to α-syn ratio for 60 min and probed for α-syn by immunoblot (C). These mock (red) and HOCl (blue) treated samples were subjected to 10 fold serial dilutions and analyzed for recombinant α-syn seeding activity (D). 20 μl per well of 10−2 through 10−4 sample dilutions were used as reaction seeds as indicated. Negative control reactions were run with no seed (gray). Other controls indicated that direct addition of 10−3 and 10−4 dilutions of BrioHOCl to the seeded polymerization reactions without preincubation with the α-syn seed had no effect on the reaction kinetics, whereas a 10−2 dilution partially interfered with the reaction (S3 Fig). Each trace represents the average ThT fluorescence of 4 replicate wells. Similar results were obtained in two additional independent experiments.