Figure 7A. Raw264.7 cells were transfected with Flag-Rab10 or control empty vector. The transfected cells were lysed in the presence of 2 mM GTPγS or 2 mM GDP. The Flag-Rab10 complex was immunoprecipitated with Flag-resin followed by extensive washing and elution of complexes with 200 μg mL-1 3xFlag-tide. Eluates were analyzed by SDS-PAGE followed by immuno-blotting with anti-Flag-HRP antibody. B. Purified complexes were digested with trypsin and peptides identified with an Orbitrap mass analyzer. A representative table of peptides matching proteins with spectral counts >10 from either the GTPγS or GDP incubations, and <5 spectral counts in the control elution. C. Flag(N-term)-Rab10 protein was purified from HEK293-FT cells transfected with Flag(N-term)-Rab10 plasmids and immobilized on Flag-resin in lysis buffer containing 2 mM GTPγS, or 2 mM GDP, as indicated. Beads were mixed with lysates from cells transfected with Myc-mkate2(N-term)-EHBP1L1 to generate Rab10:EHBP1L1 complexes. Total Rab10 on the beads was detected using anti-Flag antibody, and EHBP1L1 immobilized to total Rab10 detected with an anti-Myc antibody. Representative immunoblots from n=3 biologically independent experiments are shown. D. Quantification of relative EHBP1L1 pulled down. Data are from n=3 biologically independent experiments. Error bars represent ± SEM... E. HEK293-FT cells were transfected with Flag(N-term)-Rab10 and Myc(N-term)-LRRK2R1441C to induce Rab10 phosphorylation. Membrane bound Flag(N-term)-Rab10 protein was extracted with Flag-resin to generate 'pRab10 beads,' or extracted from cells treated with MLi2 (100 nM) to generate 'Rab10 beads.' Beads were mixed with lysates from HEK293-FT cells transfected with Myc-mkate2(N-term)-EHBP1L1 to form on-bead Rab10:EHBP1L1 complexes. The total Rab10 and pT73-Rab10 on the beads was detected using anti-Flag antibody and pT73-Rab10 antibody. The EHBP1L1 in the lysates and immobilized on the beads was detected using anti-Myc tag antibody. Representative immunoblots are shown. F. The relative EHBP1L1 pulled down, normalized to total Rab10 protein on-bead, was determined from n=3 biologically independent experiments. Error bars represent ± SEM. Each dot represents one experiment, with significance assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001. G. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 (epifluorescence shown in green) and Myc-mkate2(N-term)-EHBP1L1 (epifluorescence shown in blue), or Flag(C-term)-RILPL2 (anti-Flag signal shown in blue). Cells were stained for pT73-Rab10 (shown in red). Representative photomicrographs of individual vesicles (from >10 cells analyzed for each condition from n=3 biologically independent experiments) are shown. Scale bar represents 1 μm. H. Pearson's colocalization coefficient for Rab10 and pT73-Rab10, EHBP1L1 and RILPL2 on vesicle bodies and tubular vesicles. Data are from n=20 vesicles with tubular structures positive for pT73-Rab10, and n=12 vesicles with tubular structures positive for EHBP1L1, or RILPL2, calculated from cells from three independent experiments. Each dot represents one vesicle body or tubular structure, with significance assessed by Kruskal-Wallis test followed by Dunn's multiple comparison test with *** representing P<0.0005 and **** P<0.0001. Error bars represent ± SEM.. I. Raw264.7 cells were co-transfected with Flag(N-term)-Rab10, Flag(N-term)-LRRK2R1441C, and Myc(N-term)-EHBP1L1, with representative immunoblots shown. J. Relative pT73-Rab10 levels were calculated as fold of cells transfected with Flag(N-term)-Rab10 and Flag(N-term)-LRRK2 only. Data are from n=3 biologically independent experiments. Each dot represents one independent experiment. Significance was assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001. K. Raw264.7 cells were co-transfected with eGFP(N-term)-Rab10 (epifluorescence, shown in green) and Myc-mkate2(N-term)-EHBP1L1 or Myc-mkate2 control plasmid (epifluorescence, shown in red). Representative photomicrographs are shown. Bounding boxes highlight vesicles in "High Mag" panels. Scale bars represent 10 μm, and 1 μm for "High Mag" images. L. % of eGFP(N-term)-Rab10 vesicles positive with pT73-Rab10 were calculated (from 25 cells analyzed for each condition from n=3 biologically independent experiments). Each dot represents the mean frequency observed in one cell. Error bars show ± SEM with significance was assessed by Mann-Whitney test. *** represents P<0.0005.