HEK 293 cells were transfected with LRRK2-NanoLuc vector (Promega, NV3401) for 24h, harvested, and resuspended into OptiMEM media without phenol red (Life Technologies). The cells were then seeded into white 384well plate (Corning3570) at a density of 6000 cells/well. Digitonin solution (final concentration 50 µg/ml), testing PROTACs at decreasing concentrations (11 concentrations with 2-fold serial dilution starting from 33 µM) or DMSO, and VCB protein labeled with Bodipy 576/589 (final concentration 0.5 µM) were added separately. Each well was added with NanoBRET NanoGlo Substrate (Promega, N2160) before performing the NanoBRET reading on PHERAstar (BMG LABTECH) plate reader equipped with a 450-nm bandpass filter (donor) and a 600-nm long pass filter (acceptor). NanoBRET ratio of each well was expressed in milliBRET according to the equation: mBRET = [(signal at 610 nM/signal at 450 nM) -(signal at 610 nMno tracer control/signal at 450 nMno tracer control)]×1000. The background signal as shown in the DMSO control samples was subtracted from each sample.