Soluble LRRK2 phosphorylates plasma membrane–targeted Rab10 more efficiently than mitochondrially anchored LRRK2. (A) Representative immunoblot of HeLa cells transfected with either soluble GFP-R1441G LRRK2 (with soluble Myc-GBP; lanes 1, 2, 5, and 6) or mitochondrially anchored R1441G-LRRK2 (with mito-GBP; lanes 3, 4, and 7–10) and Myc-Rab10 (lanes 5–8) or PM-Rab10 (lanes 1–4, 9, and 10). One set of samples was treated with 200 nM Mli-2 at the time of transfection (lanes 9 and 10). Blots were probed for pRab10, pS1292 LRRK2, GFP, and Myc-tag. An ∼37-kD nonspecific band is shown below as a loading control. (B) Light microscopy of an A549 Rab10 knockout cell expressing GFP-R1441G LRRK2 (green), Myc-GBP, and PM-Rab10 (cyan). pRab10 shown in red. See also Video 2. Scale bar, 10 µm. (C) Quantitation of data shown in A. Ratio of pMyc-Rab10/total Myc-Rab10 signal normalized to the amount of total LRRK2 in the sample. Error bars represent SEM from four independent experiments. ****, P < 0.0001, unpaired t test two-tailed. (D) Quantitation of A. Ratio of phosphorylated PM-Rab10/total PM-Rab10 signal normalized to total LRRK2. Error bars represent SEM from three independent experiments. Unpaired t test two-tailed P value = ns (E) Quantitation of the percentage of A549 Rab10 knockout cells transfected with GFP-R1441G LRRK2, Myc- or mito-GBP, and phosphorylated PM-Rab10 positive. Scored based on presence or absence of phosphorylated PM-Rab10. Error bars represent SEM from four independent experiments with >30 cells scored per experiment; **, P = 0.0029, two-tailed unpaired t test.