Hpa008784

For Western blot analysis, cells were harvested with a plastic cell scraper, mixed with Laemmli buffer, and incubated at 95°C for 10 min. Proteins were then resolved on a SDS–PAGE gel at 150 V and transferred to a nitrocellulose membrane by Western blotting at 300 mA. Membrane was blocked with 2% milk in PBST and stained with anti‐FUS antibody (HPA008784, Sigma‐Aldrich) diluted 1:10,000 in 2% milk in PBST. The membrane was then stained with HRP‐conjugated secondary antibody (Sigma‐Aldrich) diluted 1:5,000 in 2% milk in PBST. Membranes were covered with the substrate Luminata Crescendo (Millipore), and chemiluminescence was detected on Amersham Hyperfilm ECL (GE Healthcare).

Equal amounts of each brain fraction were loaded into a 5–20% SDS–polyacrylamide gel (e-PAGEL®, E-T520L, Atto), and the proteins were separated and transferred to polyvinylidene difluoride membranes (Immobilon®-P Transfer Membrane, Merck). The membranes were blocked with 2.5% goat serum in Tris Buffered Saline with Tween 20 (TBST) and incubated with the following antibodies at 4 °C overnight: anti-⍺-syn (D37A6, Cell Signaling Technology), anti-tau (T57120, BD Transduction), anti-MAP2 (M9942, Sigma-Aldrich), anti-phosphorylated-synuclein (pSyn #64, Wako), anti-phosphorylated-tau (PHF13 S396, 10-444, Cell Signaling Technology; Phospho-Tau S262, #44-750G, Invitrogen; AT8 S202/T205, 00-1566, Innogenetics), anti-FUS (HPA008784, Sigma-Aldrich; 11570-1-AP, Proteintech), and anti-TDP43 (G400, Cell Signaling Technology) antibodies in 2.5% goat serum/TBST (1:1000). The membranes were washed in TBST for 10 min twice and incubated in a horseradish peroxidase secondary antibody (Immobilon® Forte Western HRP Substrate) diluted with 2.5% goat serum/TBST (1:2000) for 1 h, at room temperature. The immunoreactive proteins were visualized via the application of the substrate for enhanced chemiluminescence (Luminata, Millipore). The signals were acquired using ImageQuant LAS-4000 (GE Healthcare).