Artificially anchored, nucleotide-bound Rab29 and Rab10 are substrates for pathogenic LRRK2. (A) Light microscopy of A549 Rab10 knockout cells transfected with GFP-R1441G LRRK2 (green) and mito-Rab10 (cyan). pRab10 is shown in red. Scale bar, 10 µm. (B) HEK293T cells transfected with GFP-R1441G LRRK2 and mito-Rab10, Myc-Rab10, or Myc-Rab29 T23N. Lysates were immunoblotted for pRab10, Myc-tag, pS1292 LRRK2, GFP for total LRRK2, and tubulin. A portion of the Ponceau S–stained filter (∼25 kD) is shown as a loading control. (C) HEK293T cells transfected with R1441G LRRK2, mito-Rab29, or Myc-Rab29 expressed for 24 h. Lysates were immunoblotted for pThr71 Rab29, GFP for total LRRK2, PS1292, and Myc-tag. A portion of the Ponceau S–stained filter (∼20 kD) is shown as a loading control. (D) Quantitation of B. Error bars represent SEM from three independent experiments. P values calculated from unpaired t test. *, P = 0.02 (two tailed). (E) Quantitation of C. pRab10 or Rab29 levels normalized to total Rab and p1292/total LRRK2 levels. In D and E, error bars represent SEM from three independent experiments with duplicate samples. Two-tailed P values were calculated using an unpaired t test.