Protein Extraction and Western Blot Analysis

Protein extraction was performed using gentle hypotonic lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 2 mM EDTA, 0.5% Triton-X-100) supplemented with 1 × complete protease inhibitor (Roche). After 30 min incubation on ice, the NaCl concentration was adjusted to 150 mM, followed by another 5 min incubation on ice. The lysate was cleared by centrifugation at 16000xg for 15 min at 4 °C. Western blot analyses were performed according to the already published protocol from our laboratory^{16 (link)}. Briefly, 30 μg of proteins were separated by SDS–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), blocking was done with 5% skim milk diluted in PBS or TBS with 0.1% Tween-20 (PBS-T or TBS-T), and then incubated for 1.5–2 h at room temperature (RT) with anti-FUS (Santa Cruz, 4H11, 1:3000 dilution in PBS-T), home-made polyclonal rabbit anti-FUS^{9 (link)}, anti-actin (MP Biomedicals, 691,001, 1:25,000 dilution in PBS-T), anti-FBL (Santa Cruz, sc-25397, 1:500 dilution in TBS-T), anti-NOP56 (Santa Cruz, sc-23701, 1:500 dilution in TBS-T), anti-EWSR1 (Santa Cruz, sc-48404, 1:500 dilution in TBS-T) primary antibodies. After washing, the membrane was incubated for 1 h at RT with species-specific horseradish peroxidase (HRP)-coupled secondary antibody (Santa Cruz, sc-2005, goat anti-mouse; Santa Cruz, sc-2357, mouse anti-rabbit; Santa Cruz, sc-2020, monkey anti-goat; 1:3000 dilution in PBS-T). The signal was detected using the enhanced chemiluminescence method (ECL, GE Healthcare) and quantified using Image J software. Immunofluorescence was performed according to the protocol described in^{16 (link)}. Images were acquired using an Olympus Fluoview 1200 IX83 confocal scanning microscope with a 60 × oil-immersion objective. Two channels were used to acquire images with the following excitation parameters: 488 nm for Alexa Fluor 488 and 405 nm for DAPI. The obtained images were analyzed using ImageJ software.