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. 