Figure 5A. Raw264.7 cells were transfected with Flag(N-term)-LRRK2 and eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells with eGFP(N-term)-Rab10 epifluorescence (shown as green signal), and immuno-stained for pT73-Rab10 (shown as red signal) and LRRK2 (shown as blue signal). White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Scale bars represent 10 μm or 1 μm in "Hig Mag" panels. B. Raw 264.7 macrophage cells transfected with eGFP(N-term)-Rab10 and Flag(N-term)-LRRK2 were sequentially lysed mechanically into buffer to create a 'soluble' protein fraction, and then insoluble material lysed into triton X-100 buffer to create a "triton x-100" fraction. The "soluble" and triton X-100 buffer solubilized fractions were analyzed using SDS-PAGE (TGX gels), and representative immunoblots from n=3 independent experiments are shown. C. The "triton X-100" fraction was used as input for LRRK2 immunoprecipitation, with subsequent detection of total Rab10 and pT73-Rab10 proteins in LRRK2:Rab10 immunocomplexes using SDS-PAGE (TGX gels). To measure the stoichiometry of pT73-Rab10 to total Rab10, the same immunocomplexes were analyzed using phos-tag SDS-PAGE method, with unphosphorylated eGFP(N-term)-Rab10 migrating below the phospho-Rab10 protein. Immunoblots are representative of three independent experiments, with similar results obtained. D. Calculated levels of total LRRK2:Rab10 protein complex with and without LRRK2 kinase inhibition. Data are from n=3 biologically independent experiments with error bars showing ± SEM. Significance was assessed by a two-tailed t-test with N.S representing P>0.05. E. The signal intensities from pT73-Rab10 and unphosphorylated Rab10 in the input lysate co-immunoprecipitated with LRRK2 antibodies. The stoichiometry of pT73-Rab10 was calculated by dividing the pT73-Rab10 signal by total Rab10 signal (total Rab10 signal equivalent to pT73-Rab10 signal+ unphosphorylated Rab10 signal). Data are from n=3 biologically independent experiments with error bars showing ± SEM; Significance was assessed by two-tailed t-test with *** representing P<0.0005. F. Model for LRRK2 interaction with Rab protein substrates. GDP-bound Rab10 interacts with a GDI (Guanosine nucleotide dissociation inhibitor) and remain largely cytosolic. GEFs (guanine nucleotide exchange factors) facilitate the exchange of GDP for GTP to promote the endosome localization of Rab10. LRRK2 is temporarily recruited by GTP-bound active Rab10 on endosomes and dissociates from Rab10 after phosphorylation occurs, thereby facilitating substrate turnover. LRRK2 interaction and phosphorylation may block the interaction of Rab10 GAPs (GTPase-activating proteins) that would otherwise catalyze GTPase activity of Rab10 and promote Rab10 dissociation from endosomes (Liu et al., 2018). Without GAP interactions, Rab10 remains GTP-bound and associated with endosomes. Therefore, LRRK2 might prolong the association of Rab10 on endosomes in a GTP-bound form, but modified with phosphorylation that affects interactions with typical Rab10 effectors.