Eukaryotic expression constructs for α-synuclein, 3flag-LRRK2 or eGFP-LRRK2 overexpression are described elsewhere [34 (link),60–63 (link)]. Untagged LRRK2 was generated by BamHI restriction-mediated excision of the 3flag-tag. LRRK2 knockdown constructs were cloned according to [64 (link)] and used with a blasticidin resistance marker (as in [34 (link)]). All transgenes were cloned in the pCHMWS backbone as described in [65 (link)]. A CMV promoter was used for all cell culture applications, while a CaMKII0.4 promoter was applied for in vivo overexpression purposes. All constructs were sequence confirmed. Lentiviral (LV) vectors were produced as described in Ibrahimi et al. [66 (link)]. Anti-α- or β-tubulin, vinculin and anti-FlagM2 antibodies are purchased from Sigma-Aldrich, anti-α-synuclein antibody from Enzo Life Sciences, LRRK2 P-S935 from Novus Biologicals and MJFF-2 from Abcam. Anti-eGFP antibody is generated in-house [67 (link)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and lactate dehydrogenase (LDH)-based viability kits are purchased from Roche. LRRK2 kinase inhibitor-1 (L2-IN1) [32 (link)] was purchased from Calbiochem and PF-06447475 [68 (link)] from Sigma-Aldrich.
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