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. 2021 Mar 30;296:100613. doi: 10.1016/j.jbc.2021.100613

Figure 2.

Figure 2

ASyn-nLuc Oligomers can seed ASyn fibrillization. In total, 50 μM each of ASyn-LgBiT and ASyn-SmBiT (A) or 100 μM untagged ASyn (B) was incubated for the indicated times at 37 °C and then analyzed by size-exclusion chromatography with multiangle light scattering (SEC-MALS). Solid lines are refractive index and dotted lines are the calculated molecular weights. ASyn converts from a largely monomeric to a largely trimeric state over the incubation time course of our assays (24 h nLuc, 96 h. FRET). C–E, preformed split nLuc-tagged ASyn oligomers are used as seeds for an ASyn fibrillization assay as described in methods. nLuc-tagged ASyn oligomers are formed in a still preincubation of ASyn-LgBiT and ASyn-SmBiT and subsequently seeded into an untagged ASyn fibrillization assay. Various percentages of ASyn-nLuc oligomers in a large excess of untagged ASyn are incubated as described in methods and ASyn fibrils formed are quantified using ThT fluorescence. C, seeding capabilities increase with preincubation time for a 1% seeding. D, zero time of preincubated does not seed fibrillization. E, seeding capacity of 24 h preincubated split nLuc-tagged ASyn increases with amount of seed added. Mean ± SEM shown, n = 3.