Fig. 1: LRRK2 generates membrane reservoirs via RAB10 T73 phosphorylation.

a Current model of RAB10’s roles in STm invasion. b WT Henle 407 cells were treated with 50 nM MLi-2 for 90 min and cell lysates were immunoblotted with indicated antibodies. c and d Representative images (c) and quantifications (d) of RAB10⁺ tubules (visualized with anti-RAB10 antibody) in WT Henle 407 cells in control condition or treated with MLi-2 as in (b). e Representative images of Henle 407 cells transfected with GFP-RAB10 and stained for phospho-RAB10 (T73), with or without MLi-2 treatment. f Line plot profile of the white arrow in the inset (control) in e. Arb. units (arbitrary units) indicate the signal densities along the chosen white arrow. g and h Representative images (g) and quantifications (h) of RAB10⁺ membrane reservoirs in WT Henle 407 cells transfected with GFP-RAB10 and treated with MLi-2. Arrow indicates a RAB10⁺ membrane reservoir structure. i and j Representative images (i) and quantifications (j) of CellMask⁺ membrane reservoirs in WT Henle 407 cells in control condition or treated with MLi-2 as in (b). Cells in both conditions did not have RAB10 overexpressed. k CRISPR/Cas9-mediated deletion of LRRK2. l and m, Representative images (l) and quantifications (m) of endogenous RAB10⁺ membrane reservoirs in indicated cells in basal growth condition. n and o, Representative images (n) and quantifications (o) of RAB10⁺ membrane reservoirs in RAB10 KO Henle 407 cells transfected with myc-PM-RAB10 constructs, and treated with or without MLi-2 (50 nM, 90 min). All images shown are representative images from three independent experiments. Data shown are means ± standard deviation (S.D.) for three independent experiments. At least 100 cells for each condition in each experiment were scored for the presence of RAB10-(c, g and l) or CellMask-(h) positive tubules. P values were calculated using two tailed unpaired t-test (d, h and j), one-way analysis of variance (ANOVA) (m), or two-way ANOVA (o). Scale bars, (c, g, i, l and n) 10 μm, (e, upper and lower panels) 10 μm, (e, middle panel) 3 μm. Source data are provided as a Source Data file.