Lrrk2

DC2.4 cells or sCD83-treated DC2.4 cells were lysed with RIPA buffer (Beyotime Biotechnology, Shanghai, China). Identical quantities of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Subsequently, 5% non-fat dry milk in Tris-buffered saline 0.1% Tween 20 (TBS-T) was used to block non-specific binding sites for 1 h. After washing with TBS-T, membranes were incubated with primary antibodies against mouse Rab1a (Abcam, Cambridge, MA, USA), LRRK2 (Abcam, Cambridge, MA, USA), F-actin, MHC-II (R&D Systems, Inc., Minneapolis, MN, USA), and GAPDH (Cell Signaling Technology, Beverly, MA, USA) at 4°C for overnight. Membranes were then washed and incubated with secondary antibodies (goat-anti-rabbit IgG antibodies) conjugated to horseradish peroxidase (Beyotime Biotechnology, Shanghai, China) for 1 h. Finally, the membranes were developed using the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Densitometric analyses were performed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Protein samples were prepared from the homogenized lysates of mouse brain tissue, and the concentrations were detected with the Pierc BCA Protein Assay Kit (Invitrogen). Proteins were separated by SDS-PAGE gel (Epizyme, China), transferred to a polyvinylidene fluoride membrane, and blocked with 5% milk for 1 h at RT. The membranes were then incubated with the following primary antibodies: LRRK2 (Abcam), AQP4 (Proteintect), p-Ser (Millipore), and GAPDH (Cell Signaling Technology) overnight at 4 °C and a secondary antibody (Cell Signaling Technology) for 1 h at RT. Finally, the membrane was visualized using a chemiluminescence method. All blots or gels derive from the same experiment and they were processed in parallel.

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).