Figure 6. Knock-out of Rab29 does not reduce elevated Rab10 phosphorylation in pathogenic LRRK2[R1441C] knock-in mice.

(A) The indicated matched primary MEFs were treated with vehicle (DMSO) or 100 nM LRRK2 inhibitor MLi-2 for 90 min prior to harvest. Twenty micrograms of whole-cell extracts were subjected to quantitative immunoblot analysis with the indicated antibodies. Technical replicates represent cell extract obtained from a different dish of cells. Cell extract derived from a wildtype MEF cell line (WT*) was added to each gel in order to accurately compare the Rab10 pThr73/Rab10 total ratios between genotypes. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Quantified data are presented as the ratios of phospho-Rab10/total Rab10, calculated using the Image Studio software. Values were normalized to the average of wildtype MEFs treated with DMSO. Similar results were obtained in two separate experiments. Quantifications are presented as mean ± SD. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons test and there was a statistically significant difference between wildtype and LRRK2[R1441C] knock-in MEFs (****P < 0.0001) but not between wildtype and Rab29 knock-out MEFs (P = 0.8351) or between LRRK2[R1441C] knock-in and Rab29 knock-out LRRK2[R1441C] knock-in MEFs (P = 0.9452). (B–D) The indicated 6-month-old matched mice were administered with vehicle (40% (w/v) (2-hydroxypropyl)-β-cyclodextrin) or 30 mg/kg MLi-2 dissolved in vehicle by subcutaneous injection 2 h prior to tissue collection. Forty micrograms of tissue extracts were subjected to quantitative immunoblot analysis with the indicated antibodies. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Quantified data are presented as the ratios of phospho-Rab10/total Rab10 and phospho-Rab12/total Rab12, which were calculated using the Image Studio software. Quantifications are presented as mean ± SD, normalized to vehicle treated, wildtype animals. Each lane represents a tissue sample from a different animal. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons test and there was a statistically significant difference in Rab10 phosphorylation between wildtype and LRRK2[R1441C] spleen samples (**P = 0.0093 (E)). All other phospho-Rab10 comparisons were not statistically significant. Wildtype vs LRRK2[R1441C]: P = 0.7169 (B), P = 0.2792 (C), P = 0.1555 (D). Wildtype vs Rab29 knock-out: P = 0.2594 (B), P = 0.8942 (C), P = 0.9372 (D), P = 0.3964 (E). LRRK2[R1441C] vs Rab29 knock-out LRRK2[R1441C]: P = 0.6629 (B), P = 0.9955 (C), P = 0.9962 (D), P = 0.4184 (E). There was a statistically significant difference in Rab12 phosphorylation between wildtype and LRRK2[R1441C] tissue samples: brain ****P < 0.0001 (B), lungs *P = 0.0438 (C), kidneys **P = 0.0041 (D) and spleen *P = 0.0390 (E). All other phospho-Rab12 comparisons were not statistically significant. Wildtype vs Rab29 knock-out: P = 0.3057 (B), P = 0.8915 (C), P = 0.9595 (D), P = 0.2078 (E). LRRK2[R1441C] vs Rab29 knock-out LRRK2[R1441C]: P = 0.7791 (B), P = 0.9747 (C), 0.6681 (D), P = 0.4093 (E).