Mitochondrially anchored Rab29-GTP activates LRRK2 as efficiently as Golgi-associated Rab29. (A) Quantitation of Fig. 3 for HeLa (left) and RPE cells (right). Pearson’s correlation coefficients calculated from ≥20 cells are shown. Error bars represent SEM. (B) HEK293T cells were transfected with LRRK2-R1441G and either Myc-Rab29 or mito-Rab29, Rab9, or mito-Rab29 D63A. Samples were immunoblotted for pS1292 LRRK2, total LRRK2 (UDD3), and Myc-tag after 24 h of expression. An ∼50-kD nonspecific band is shown at left as a loading control; a portion of the Ponceau S–stained filter (∼25 kD) is shown at bottom right as a loading control. (C) Quantitation of left panel of B. Amount of active (pS1292) LRRK2 normalized to the amount of total LRRK2 and Rab29. Error bars represent SEM from four independent experiments; P > 0.5 from two-tailed unpaired t test. (D) Quantitation of right panel of B. Amount of active, pS1292 LRRK2 normalized to the amount of total LRRK2 and Rab29. Error bars represent SEM from three experiments with duplicate samples in each; P values from unpaired t test: **, P = 0.0017; ****, P < 0.0001. (E) Immunoblot of membrane proteins and the equivalent volumes of cytosolic proteins from HEK293T cells transfected with Myc-Rab29 WT, Myc-Rab29 D63A, mito-Rab29 WT, or mito-Rab29 D63A. Blots were probed with anti-Myc antibodies for Myc-Rab29 or mito-Rab29 anti-LAMP2 as a membrane marker and anti-tubulin as a cytosolic marker.