Hpa013144

Soft tissues and periosteum were removed from mice femora by microsurgery forceps under the dissecting microscope. Diaphyses were extracted, and bone marrow was flushed away. Femoral diaphyses were then crushed using a mortar and pestle in liquid nitrogen. Both bone and cellular proteins were extracted by incubating in radioimmunoprecipitation assay lysis buffer containing protease inhibitor (Roche) and a phosphatase inhibitor cocktail (Sigma-Aldrich) for 30 min at 4°C. Clarified bone and cell lysate (obtained by centrifugation at 16,000g for 20 min at 4°C) were diluted with 4× SDS sampling buffer and boiled for 5 min. For each sample, 4 μg of protein was fractionated on 10 to 17.5% SDS–polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane (Millipore). Membranes were incubated with primary antibodies, including β-actin JLA20 antibody (1:5000; Developmental Studies Hybridoma Bank), Mfn2 antibody (1:1000; 9482, Cell Signaling Technology), or VAPB antibody (1:1000; HPA013144, Sigma-Aldrich), and the corresponding horseradish peroxidase–conjugated secondary antibodies. Proteins were visualized by enhanced chemiluminescence and autoradiography (FujiFilm LAS-3000/4000 Gel Documentation System, Japan).

Four‐micrometer‐thick sections of the basal ganglia, midbrain, pons, medulla oblongata, and cerebellum were cut and subjected to immunohistochemical processing using the avidin‐biotin‐peroxidase complex (ABC) method with a Vectastain ABC kit (Vector, Burlingame, CA, USA). Antibodies against phosphorylated α‐Syn (p‐α‐Syn) (#64; Wako, Osaka, Japan; 1:5000) and VAPB (HPA013144; Sigma, St. Louis, MO, USA; 1:250) were used as primary antibodies. The sections were pretreated in an autoclave for 10 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. For p‐α‐Syn immunohistochemistry, the sections were additionally pretreated in 98% formic acid for 5 min. Diaminobenzidine was used as the chromogen. The sections were counterstained with hematoxylin.