Bioluminescence Split Complementation Assay for α-Synuclein

Bioluminescence split complementation assay with the different mutants was performed using α-SYN-hGLuc1 (S1) and α-SYN-hGLuc2 (S2) constructs based on the principle reported in the publication [59 (link), 64 (link)]. Briefly, equal numbers of α-SYN knock out HEK293T cells were seeded in 24-well plate in triplicates. For each well, 500 ng of the individual α-SYN containing split luciferase constructs (S1 + S2) were transfected along with 10 ng of the WT or mutant α-SYN constructs in a 1:100 ratio. At each time point, the media was assayed for luciferase activity using BioLux® Gaussia Luciferase Assay Kit (NEB# E3300S) following the manufacturer’s protocol.