Figure 4.

LRRK2 kinase inhibition attenuates inflammatory signaling in microglia. A, B, Calculation of the inhibitory potential of the LRRK2 targeted compounds L2in1 (A) and sunitinib (B) using standardized in vitro kinase assays consisting of 30 nm LRRK2 enzyme, 50 μm peptide, and 100 μm ATP with reactions run for 30 min. IC₅₀ values were calculated through nonlinear regression with r² values of 0.960 and 0.968, respectively, for L2in1 and Sunitinib. C, Graphical depiction of the experimental timeline used to generate lysates and serum from primary microglia analyzed in D–G. D, Quantification of secreted TNFα by ELISA after a 6 h exposure to LPS. Suni, Sunitinib. Drug concentrations are given in micromoles, and mean values are calculated from three independent experiments. E, TNFα mRNA was measured by quantitative PCR (ΔΔcT) normalized to TBP, and mean values are calculated from three independent experiments. F, Representative Western blot analysis of primary microglia lysates. G, Quantification of three independent experiments with levels of iNOS and phospho-p38 levels normalized to VDAC expression. *p < 0.05, **p < 0.01, one-way ANOVA with Tukey–Kramer test, with respect to DMSO (+LPS) conditions. Error bars indicate SEM.