LRRK2 and RAB Transfection in HEK293T and SH-SY5Y Cells

HEK293T cells were cultured as described (Madero-Perez et al., 2018 (link)) and transfected at 80% confluence with 2 μg of LRRK2 constructs and 200 ng of RAB constructs where indicated, and 6 μl of LipoD293 (SignaGen Laboratories) per well of a 6-well plate for 5 h in full medium, resulting in a roughly 30% transfection/co-transfection efficiency, respectively. Cells were split 1:5 the following day, and processed for immunocytochemistry or Western blotting 48 h after transfection.
SH-SY5Y cells stably expressing GFP, flag-tagged wildtype LRRK2, or flag-tagged G2019S-mutant LRRK2 were cultured as described (Madero-Perez et al., 2018 (link)) and subcultured at a ratio of 1:6 twice a week. Transfection of cells was carried out at 80% confluence with 0.4 μg of DNA and 1.5 μl of Lipofectamine 2000 (Invitrogen) per well of a 24-well plate in 200 μl OptiMEM. Five hours later, cells were changed into full medium, passaged the following day at a 1:5 ratio onto coverslips, and fixed and stained 72 h after transfection.
Where indicated, cells were treated with brefeldin A (7.5 μg/ml, Sigma-Aldrich) or with nocodazole (200 nM, Sigma-Aldrich) for 3 h, or with 100 nM MLi2 (MRC PPU, Dundee, United Kingdom), 500 nM LRRK2-IN1 (obtained through the MJFF) or 500 nM GSK2578215A (Tocris) for 1 h before fixation.