HTRF Human and Mouse Total LRRK2 Detection Kit, 10,000 assay points

The Total LRRK2 assay quantifies the expression level of LRRK2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total LRRK2 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of LRRK2 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample and provides a means of assessing the protein's expression under a no-wash assay format.

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of HTRF Total LRRK2 detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Detection of Total LRRK2 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol allows miniaturization while maintaining robust HTRF quality.

As expected, the warhead HG-10-102-01 did not induce any signal decrease, while PROTAC XL01126 dose-dependently degraded LRRK2. This degradation was blocked in the presence of Epoxomicin. In this experiment, the overnight treatment with the PROTAC yielded the best DC50 (4.5 times better than that obtained after a 4-hour treatment). The highest degradation rate (Dmax) was approximately 70% and was achieved with 1 µM of PROTAC. These results were further supported by the ATPlite 1step viability assay, where no cytotoxic effects were observed for the different compounds (data not shown here)

HEK293 cells were seeded in a 96-well culture plate at 12,500 cells/well and transfected with a plasmid encoding the human mutant LRRK2 G2019S protein. The human lung cancer cell line A549 and the mouse fibroblastic cell line NIH-3T3 were seeded at 100,000 and 200,000 cells/well, respectively, in a 96-well culture plate. After 24 hours of incubation, the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at room temperature under gentle shaking.

HEK293 cells were transfected in a T175 flask with a plasmid encoding the human mutant LRRK2 G2019S protein for 24 hours, and then treated with 100 nM Calyculin-A for 30 minutes. The cells were lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at room temperature under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of HTRF Total LRRK2 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Total LRRK2 assay, 100 cells/well were enough to detect a significant signal, while 400 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, under these conditions, the HTRF Total LRRK2 assay was four times more sensitive than the Western Blot technique.

Under physiological conditions, LRRK2 activation and localization are regulated by the Rab GTPase Rab29. Rab29 recruits LRRK2 to the membranes of different organelles (trans-Golgi network, mitochondria, phagosomes, and damaged lysosomes) under specific stimuli. This recruitment leads to the stimulation of LRRK2 kinase activity and the subsequent increase in LRRK2 autophosphorylation at Ser1292. Activated LRRK2 phosphorylates a subset of membrane-associated Rab GTPases (Rab3, Rab8, Rab10, Rab12, Rab35, Rab43) and interacts with other downstream proteins, which together regulate a variety of cellular processes such as endocytosis and vesicular trafficking, microtubule dynamics, the autophagy-lysosomal pathway (ALP), Golgi and mitochondrial function, as well as α-synuclein homeostasis.