Western blot analysis

Five million cells were pelleted and resuspended in 100 µl of lysis buffer consisting of 1% Triton X-100, 50 mM Trizma-HCl, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail (Sigma-Aldrich, P8340), and Halt phosphatase inhibitor cocktail (Thermo Fisher, 78420). After a 10-min incubation on ice, lysates were spun at 10,000×g and the supernatant was collected. The protein was quantified using the DC protein assay (Bio-Rad, 5000112). Due to differing endogenous LRRK2 levels, 40 µg of ND2559, 60 µg of ND264, or 100 µg of ND11, ND1618, ND312, ND2752 sample were incubated at 100 °C for 5 min with NuPAGE Sample loading dye (Thermo Fisher, NP0007) and dithiothreitol as reducing agent. After SDS-PAGE, the blots were blocked in 5% w/v nonfat dry milk in 1X PBST (0.05% Tween 20). For our investigations, the following primary antibodies were used: rabbit anti-LRRK2 c41-2 (Abcam, ab133474, 1:2000), mouse anti-LRRK2 N241A/34 (Antibodies Inc., 75-253, 1:2000), rabbit anti-LRRK2 pS935 (Abcam, ab133450, 1:2000), rabbit anti-LRRK2 pS955 (Abcam, ab169521, 1:2000), rabbit anti-LRRK2 pS973 (Abcam, ab181364, 1:2000), mouse anti-Rab10 (Abcam, ab104859, 1:1000), rabbit anti-Rab10 pT73 (Abcam, ab230261, 1:1000), mouse anti-Rab8a (Novus, H00004218-M02, 1:2000), rabbit anti-Rab8a pT72 (Abcam, ab230260, 1:1000), mouse anti-β-actin (Novus, 8H10D10, 1:10,000). The blots were then probed with fluorescent-labeled secondary antibodies, IRDye donkey anti-mouse and anti-rabbit at 1:10,000 (LI-COR, 926-32212, 926-32213, 926-68072, 926-68073), and scanned using an Odyssey Imaging scanner (LI-COR). Fluorescent intensities were quantified using ImageStudio Lite software (LI-COR), and the signal from the protein of interest was normalized to the fluorescent intensity of either LRRK2 or β-actin. Values were averaged from at least three technical replicates within a cell line, and three biological replicates (three control lines and three PD cell lines). Of note, we attempted to evaluate LRRK2 Ser910 levels (Abcam, ab133449), yet this was not feasible due to a plethora of non-specific cross-reacting bands. We also attempted to measure LRRK2 Ser1292 levels by western blot using the commercially available antibody (Abcam ab203181). Using standard methods, we were unable to detect endogenous levels of LRRK2 Ser1292 in either controls or LRRK2 G2019S patient-derived LCLs.