Journal: Science Advances
Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2
doi: 10.1126/sciadv.adt2050
Figure Lengend Snippet: ( A and B ) Dose-response curve of RN277 (A) and RN341 (B) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a versus non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341. ( C ) Representative immunoblot from 293T cells transiently co-transfected with LRRK2 and GFP-Rab8a, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( D ) Sample from (C) run separately under identical conditions and immunoblotted for phospho-S935 LRRK2 and GAPDH. ( E ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from three independent immunoblots (C). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0049, DMSO versus MLi-2; *** P = 0.0004, DMSO versus Ponatinib; *** P = 0.0006, DMSO versus 5 μM RN277; *** P = 0.0003, DMSO versus 10 μM RN277; * P = 0.0406, DMSO versus 5 μM RN341; ** P = 0.0065, DMSO versus 10 μM RN341; error bars ± SEM. ( F ) Quantification of the pS935 LRRK2/LRRK2 ratio (run under identical conditions on separate blots) from three independent immunoblots (D). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **** P < 0.0001 for all conditions versus MLi-2; error bars ± SEM. ( G ) Representative immunoblot from 293T cells transiently co-transfected with GFP-Rab8a and either GFP-11 tagged wild-type (WT) or GFP-11 tagged G2019S LRRK2, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( H ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from four independent immunoblots (G). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0077, WT LRRK2 DMSO versus MLi-2; * P = 0.0324, WT LRRK2 DMSO versus 5 μM RN277; * P = 0.0461, WT LRRK2 DMSO versus 5 μM RN341; **** P < 0.0001 for all inhibitor treatments versus G2019S LRRK2 DMSO; error bars ± SEM.
Article Snippet: The Rab8A substrate, encoding a TEV cleavable N-terminal His 6 -tag (RRID: Addgene_228880; table S4) was expressed in Escherichia coli Rosetta cells (Novagen, 70954; www.sigmaaldrich.com/US/en/product/mm/70954?msockid=2313305006bb6050160c228007f861f2 ; EMD_BIO-70954).
Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection
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