Rab10

The collected cells were lysed with lysis buffer containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton x-100, and protease inhibitors. Total protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce). Cell extracts (2–3 mg) were combined with rabbit anti-FAM49B antibodies for 2 h at 4 °C followed by a second incubation with protein G Plus-agarose for 2 h and subjected to 10% SDS-PAGE followed by transfer to PVDF membranes for immunoblotting. Membranes were blocked in 5% milk/TBS-Tween for 1 h at room temperature. The membranes were incubated overnight with the primary antibody (FAM49B, 1:300, BIOSS; Rab10, 1:1000, Abcam; TLR4, 1:1000, Abcam; ELAVL1, 1:500, Abcam) at 4 °C, followed by incubation with the secondary antibody for 2 h at room temperature. Proteins were visualized using enhanced chemiluminescence (Amersham), and the results were quantified using ImageJ (NIH).

The following antibodies were used in this study: mouse monoclonal antibodies against ß-actin (Merck) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit monoclonal antibodies against MFN2 (Abcam, Tokyo, Japan), LRRK2 (Abcam), LRRK2 pS935 (Abcam), RAB10 (Abcam), RAB10 pT73 (Abcam), and TOMM20 (Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit donkey IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Alexa Fluor 488-conjugated anti-rabbit goat IgG and Alexa Fluor 555-conjugated anti-mouse goat IgG antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (MLi-2) was purchased from Cayman Chemical Co (Ann Arbor, MI, USA).