Flag lrrk2 g2019s

GSK3357679A [25 (link)]was synthesized at GlaxoSmithKline. MLi-2 was synthesized by Natalia Shpiro (University of Dundee) as described previously [16 (link)]. GZD-824 (#21508) and Rebastinib (#21465) were purchased from Cambridge Bioscience and Ponatinib (#4274) from Tocris. Microcystin-LR was purchased from Enzo Life Sciences (#ALX-350-012-M001). Human recombinant full length wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) were from ThermoFisher Scientific. Diisopropylfluorophosphate was from Sigma (#D0879).

Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific. Peptide kinase assays, performed in triplicate, were set up in a total volume of 25.5 µl containing 13.7 ng LRRK2 kinase in 50 mM Tris–HCl, pH 7.5, 0.1 mM EGTA, 10 mM MgCl₂, 20 µM Nictide (an optimized peptide substrate for LRRK2 [53 (link)]), 0.1 µM [γ-³³P]ATP (∼500 cpm/pmol) and the indicated concentrations of inhibitor dissolved in DMSO. After incubation for 30 min at room temperature, reactions were terminated by harvesting the whole reaction volume onto P81 phosphocellulose filter plate and immersion in 50 mM phosphoric acid. Samples were washed extensively and the incorporation of [γ-³³P]ATP into Nictide was quantified by Cerenkov counting using a Tri-Carb 4910 TR PerkinElmer liquid scintillation analyzer. IC₅₀ values were calculated with GraphPad Prism using non-linear regression analysis.