Pf 06447475

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.

LRRK2 kinase inhibitors CZC-25146 and PF-06447475 and casein kinase 1 inhibitor IC261 were purchased from Sigma-Aldrich. Casein kinase 1 inhibitor PF-670462 was purchased from Abcam. LRRK2 kinase inhibitor MLi-2 was kindly provided by Dr. D. Alessi (Division of Signal Transduction Therapy, University of Dundee). pCHMWS_3Flag_LRRK2_Ires_Hygro constructs of pathogenic (R1441C/G, Y1699C, and I2020T) LRRK2 variants were cloned as described in [42 (link)]. The pCHMWS_3Flag_LRRK2_Ires_Hygro constructs encoding truncated variants PLRCKW and APLRCKW, as well as the LRRK2 S908A/S910A/S935A/S955A/S973A/S976A or S908E/S910E/S935E/S955E/S973E/S976E were generated using gBlock® Gene Fragments (IDT) and as described in [42 (link)]. The following antibodies were used: mouse anti-FlagM2 (Sigma-Aldrich, F1804), mouse anti-vinculin (Sigma-Aldrich, V9131), mouse anti-α-tubulin (Sigma-Aldrich, T5168), rabbit anti-LRRK2 P-S935 (Abcam, ab133450), rabbit anti-LRRK2 P-S1292 (Abcam, ab203181), rabbit anti-LRRK2 MJFF-2 antibody (Abcam, ab133474), mouse anti-LRRK2/Dardarin, N-terminus N138/6 (Neuromab 75-188), mouse anti-LRRK2/Dardarin, C-terminus N241A/34 (Neuromab 75-253), mouse anti-LRRK2 MC.028.83.76.242 (ab130277).

LRRK2 kinase inhibitor-1 (L2-IN1), HG 10-102-01 and CZC-25146 were purchased from Calbiochem, GSK2578215A from Tocris and PF-06447475, MG132 and chloroquine from Sigma-Aldrich. MLi-2 was kindly provided by Dr. D. Alessi (Division of Signal Transduction Therapy, University of Dundee). Antibodies used are as follows: anti-flagM2, anti-vinculin and anti-α or β-tubulin (Sigma-Aldrich), anti-LRRK2 MJFF-2, anti-LRRK2 P-S935, anti-LC3 (Novus Biologicals) and anti-GAPDH (Abcam). QPCR probes were from Sigma-Aldrich, QPCR primers from IDT. Lentiviral vectors encoding full length LRRK2 were produced by the Leuven viral vector core (https://gbiomed.kuleuven.be/english/research/50000715/laboratory-of-molecular- virology-and-gene-therapy/lvvc).