HEK293 cells (ATCC #CRL-1573) cultured in complete growth medium: DMEM (Thermofisher Scientific #11960-044) supplemented with 10% Fetal Calf Serum, qualified, Brazil (Thermofisher Scientific #10270-106), penicillin/streptomycin (Thermofisher Scientific #15140-122) and L-glutamine (Thermofisher Scientific #25030-024).
N-terminus Flag-tagged LRRK2 wild-type or mutant cDNA and Flag-empty vector cDNA (negative control) in a pCMV5 vector. All plasmids used for our studies are available from the MRC PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk). These include Flag-tagged wild-type LRRK2 (DU6841), G2019S LRRK2 (DU10129), Y1699C LRRK2 (DU26486), I2020T LRRK2 (DU13081) and G2385R LRRK2 (DU27381). Polyethylenimine (PEI) “Max” (Linear, Mw 40,000) (Polysciences, Inc., #24765); 1 mg/ml (w/v) solution in milliQ water, pH 7.4; sterile filtered.
Opti-MEM Reduced Serum Medium (ThermoFisher Scientific #31985062).
Ibidi µ-Plates (24 Well) - Black ID 14 mm, ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized (Ibidi #82426).
Tissue culture-treated flat bottom cell culture 24-well plates (Thermo Scientific Nunc #142475).
4% paraformaldehyde (PFA, Sigma Aldrich #P6148) in PBS, pH 7.4 (make fresh or keep frozen at -20oC until use)
Phosphate buffered saline (PBS), pH 7.4 (ThermoFisher Scientific #10728775)
NP-40 alternative (Merck #492016)
Bovine Serum Albumin (BSA) (Sigma-Aldrich #A7906).
Primary antibodies: Monoclonal anti-Flag M2 antibody (Sigma Aldrich #F1804); anti-β-tubulin antibody (Abcam ab6046)
DAPI (bisBenzimide H 33342 trihydrochloride) (Sigma Aldrich #B2261)
Secondary antibodies: goat anti-mouse Alexa Fluor 488 (ThermoFisher Scientific #A-11029) and goat anti-rabbit Alexa Fluor 594 (ThermoFisher Scientific #A-11012)
DMSO
Lysis buffer: