Fig. 1

Altered FAM21 binding and LRRK2 kinase inhibitor-reversible Rab-GTPase phosphorylation in VKI mouse brain. A Co-immunoprecipitations of VPS26 and FAM21 with VPS35 from VKI whole-brain lysate run on a WES capillary-based western blot system (i) uncovered no effect on VPS26 pulled by VPS35 (ii, 1-way ANOVA p = 0.44) but a significant reduction in FAM21 pulled by mutant VPS35 (iii, 1-way ANOVA p < 0.003; Uncorrected Fisher’s LSD Het **p < 0.01; Ho **p < 0.01). B Western blot of Rab10, Rab10 pT73, and β-actin in VKI whole brain lysate (i) revealed no significant genotype effect on Rab10 levels (ii, Kruskal–Wallis p = 0.16), but a significant increase in Rab10 pT73 in VKI (iii, Kruskal–Wallis p < 0.0001; Uncorrected Dunn’s Het **p < 0.003, Ho **p < 0.003). C Western blot of LRRK2, LRRK2 pS935, GAPDH, Rab10, and Rab10 pT73 in VKI whole brain lysate (i) revealed that MLi2 treatment significantly reduced LRRK2 pS935 in all genotypes (ii, 2-way ANOVA treatment p < 0.0001; Uncorrected Fisher’s LSD WT-WTMLi2 ****p < 0.0001; Het-HetMLi2 ***p < 0.0002; Ho-HoMLi2 ****p < 0.0001) and had significant genotype and treatment effects on Rab10 pT73 due to significant reductions in homozygous cells (iii, 2-way ANOVA genotype p = 0.04; treatment p < 0.009; Uncorrected Fisher’s LSD WT-WTMLi2 p = 0.82; Het-HetMLi2 p = 0.10; Ho-HoMLi2 **p = 0.007). D Western blot of LRRK2, LRRK2 pS935, GAPDH, Rab10, and Rab10 pT73 in whole brain lysate revealed no significant effect of the solubilizing agent Captisol on LRRK2 pS935 (ii, Mann–Whitney p = 0.89) or Rab10 pT73 (iii, Mann–Whitney p = 0.69). For C.ii–iii, WTCap n = 5, WTMLi2 n = 6, HetCap n = 6, HetMLi2 n = 6, HoCap n = 5, HoMLi2 n = 5