Fig. 1.

Long-term LRRK2 kinase inhibitor treatment induces a sustained reduction in LRRK2 protein and phosphorylation levels, which can be completely restored after inhibitor removal. SH-SY5Y cells overexpressing 3Flag-LRRK2 WT were treated for indicated terms with CZC-25146 (200 nM), PF-06447475 (150 nM), or DMSO as control. Cell lysates were taken (a) on different days during several weeks of treatment or (b) 24 h or 72 h after removal of the LRRK2 kinase inhibitor and analyzed with immunoblotting using the FlagM2 antibody for LRRK2 detection, anti-LRRK2 P-S935, anti-LRRK2 P-S1292, anti-α-tubulin, or anti-vinculin for checking equal loading. Representative blots are shown. (a, c) Graphs show the quantification of blots representing the ratio of total LRRK2 over housekeeping proteins or the ratio of phosphorylation at S935 over total LRRK2 signal. Error bars indicate S.E.M. with N ≥ 3. Statistical significance was tested using (a) a 2-way ANOVA test with Bonferroni post-tests or (c) a Mann-Whitney U test. Triple asterisks indicate p < 0.001, double asterisks indicate p < 0.01