Anti lrrk2 mjff2

For immunoprecipitation, SH-SY5Y cells transfected for 48 hours were lysed with Tris-buffered saline containing 0.5% Triton-X, 1 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich). The lysates were rotated at 4 °C for 1hr followed by centrifugation at 20,000 g for 5 min. The supernatant was added to 30 μl (slurry volume) of Dynabeads protein G (Invitrogen) preincubated without (preclear) and with indicated antibodies and the mixture was rotated for 30 min at 4 °C. The precipitated complexes were washed three times with ice-cold PBS and then eluted with SDS sample buffer for 10 min at 90 °C. SDS-PAGE and Western Blotting were performed with NuPage Bis-Tris Mini Gel and Xcell II Blot Module (Invitrogen) according to manufacturer’s protocols. Antibodies used include: anti-FLAG M2 (Sigma-Aldrich, 1:1000), anti-V5 (Invitrogen, 1:1000), anti-Actin (C4, Abcam, 1:2000), anti-LRRK2 (MJFF2, Epitomics, 1:1000) and appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearch, 1:2000). Blots were visualized using Supersignal luminol substrate (Thermo Scientific #34075).

Freshly prepared lysis buffer (1% Triton X-100, 1X complete protease inhibitor Cocktail [Roche], and 1X Phosphatase inhibitor cocktail [Roche] in PBS) were used to homogenize whole brains or dissected brain regions (cortex [CTX], hippocampus [HC], striatum [STR], brain stem [BS] and midbrain [MB]). The protein lysates were kept on ice for 1h with occasional shaking, centrifuged for 15 min at 14.000 rpm and 4°C, and protein concentration was measured using the BCA protein Assay Kit (Pierce Thermo Scientific) following the manufacturer’s instructions. Samples were loaded on 5, 6, or 7% acrylamide SDS-PAGE or NuPAGE 3–8% Tris-Acetate precast gels (Invitrogen). After protein transfer onto polyvinylidenfluorid (PVDF) membranes (Millipore) overnight at 4°C, membranes were blocked for 1h at RT in 5% milk powder in Tris-buffered saline and 0.1% Tween-20 and incubated overnight at 4°C with primary antibody anti-MID LRRK2, anti-LRRK2 Novus-267 (Novus Biologicals), anti-LRRK2 MJFF2 (Clone c41–2) and MJFF5 (Clone c68–7) (Epitomics), anti-ß-tubulin III or anti-Vinculin (Sigma-Aldrich). Horseradish peroxidase-conjugated secondary antibodies anti-mouse (Jackson Immunoresearch) or anti-rabbit (Dako) and chemiluminiscence HRP substrate (Immobilion Western HRP substrate) were used for detection.