Mitochondrially anchored Rab29 recruits pathogenic LRRK2, pRab10, and total Rab10 to mitochondria. (A) Top row: Light microscopy of HeLa cells transfected with Myc-Rab29 (cyan) and GFP-R1441G LRRK2 (green); the TGN marker GCC185 is shown in red. Second row: Light microscopy of HeLa cells transfected with mito-Rab29 (cyan) and GFP-R1441G LRRK2 (green) expressed for 24 h. Endogenous pRab10 is shown in red. Third row: Mito-Rab29 (cyan) and GFP-R1441G LRRK2 (green); endogenous total Rab10 is shown in red. Fourth row: Mito-Rab29 (cyan) and GFP-R1441G LRRK2 (green). 24 h after transfection, cells were treated with 200 nM MLi-2 for 45 min, fixed, and stained. Endogenous pRab10 is shown in red. Fifth row: Mito-Rab29 (cyan) and GFP-R1441G LRRK2 (green); 24 h after transfection, cells were treated with 200 nM MLi-2 for 45 min, fixed, and stained. Endogenous total Rab10 is shown in red. Reference image of Rab10 without R1441G LRRK2 or mito-Rab29 expression is shown. Scale bars, 10 µm. (B) Immunoblot of HEK293T cells expressing mito-Rab29, 1441G LRRK2, and GFP-Rab10 for 24 h. Blot was probed for pRab10, Myc-tag, GFP, pS1292 LRRK2, and total LRRK2 (UDD3). MLi-2 treatment was 200 nM at the time of transfection. A portion of the Ponceau S–stained filter (∼25 kD) is shown as a loading control.