Western Blot Analysis of Protein Extracts

Western blot analysis was performed as previously described [22 (link)]. Briefly, protein extracts were prepared by direct lysis in Laemmli buffer or NP40 1% buffer when protein content was determined using the Bradford protein assay. Equal amounts of protein extracts were resolved by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently electroblotted into nitrocellulose membrane (Thermo Fisher Scientific). Membranes were incubated with 3% low-fat milk in 1X PBS-Tween 0.05% solution with the indicated antibody: anti-LRRK2 (1:5000 MJFF2 c41-2 ABCAM, Cambridge, UK), anti-Myc (1:5000 M4439 Sigma-Aldrich), anti-Flag (1:2500 F3165 Sigma-Aldrich), anti-beta-actin (A5441 1:5000 Sigma-Aldrich), anti-Sec8 (1:1000 610659 BD Biosciences, San Jose, CA, USA), anti-Exo70 (1:1000 HPA022840 Sigma-Aldrich), anti-Sec6 (1:1000 SAB2100729 Sigma-Aldrich) for 16 h at 4 °C. Goat anti-mouse immunoglobulin G (IgG) peroxidase-conjugated antibody (1:2500 Merck, Darmstadt, Germany) or goat anti-rabbit IgG peroxidase-conjugated antibody (1:5000 Millipore Corporation) were used to identify immunocomplexes by enhanced chemiluminescence (ECL start, Euroclone SpA, Milano, Italy).