Fig. 4.

Modulation of in vitro kinase activity by LRRK2-targeting Nbs. (A) Effect of Nbs on LRRK2 kinase activity measured using the LRRK2-optimized AQT0615 peptide as substrate. (B) Effect of Nbs on LRRK2-mediated phosphorylation of Rab8a determined via a Western blot assay (SI Appendix, Fig. S10). In both A and B, the influence of the Nbs (25 µM) on the relative kinase activity compared to the “No-Nb” control is plotted, and a positive MLi-2 control is included. Each bar reflects the average (±SD) of three independent measurements. (C–E, Upper) Dose–response curves for the inhibition of the in vitro LRRK2 kinase activity by the group1 Nbs: Nb1 (C), Nb6 (D), and Nb23 (E), using a serial dilution of the Nb and a fixed concentration of AQT0615 peptide (10 µM) and ATP (1 mM). (Middle) The Michaelis–Menten curves obtained for LRRK2 at varying concentrations of ATP and a fixed (subsaturating) concentration of peptide substrate (AQT0615) and at varying concentrations of the respective Nbs. (Lower) The corresponding linearizations according to the Lineweaver–Burk method (double-reciprocal plot). The Nb concentrations used are indicated below the plots. Each datapoint reflects the average (±SE) of three independent measurements. The IC₅₀ (±SD) values resulting from fitting on a three-parameter logistic equation and the K_i^app and α values (±SD) resulting from global fitting on a mixed-type inhibition mechanism are indicated on the graphs.