Mjff2

The cell lysates were centrifuged at 16,000 × g for 10 min at 4 °C to remove insoluble material. The supernatants were precleared, then immunoprecipitated with anti-p53 or anti-LRRK2 (MJFF2, Abcam) at 4 °C for 8 h, and further incubated with protein-A agarose (Pierce, Rockford, IL,USA) for 24 h. The antibody-protein complexes were subjected to Western blot analysis using the indicated antibodies.
Cell nuclei were fractionated using a Nuclear/Cytosol Fractionation kit (K266-100, Bioscience, Milpitas, CA, USA) as suggested by the manufacturer, after the indicated treatment.
G2019S TG mice (#009609, Jackson Laboratory, Bar Harbor, ME, USA) and normal control littermates were sacrificed by cervical dislocation. Brain tissues were disrupted using a Dounce Homogenizer (10 strokes) and lysed by passing the extracts through a 22-gauge needle five times. The brain lysates were centrifuged at 16,000 × g for 30 min at 4 °C to remove insoluble material, and supernatants were used for further Western blot analysis.
The immunoprecipitated antibody-protein complexes, cell or brain lysates, or fractionated samples were subjected to Western blot analysis using the indicated antibodies.

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.