Ne per 78833

SDS-PAGE, immunoprecipitation, and western blot assays were carried out as described previously (Tanigawa et al. 2013 (link); Pan et al. 2010 (link)). In brief, cell lysates were centrifuged at 500 × g for 5 min at 4 °C to prepare nuclei and cytosol by using the nuclear and cytoplasmic extraction reagents, respectively (NE-PER; 78833; Thermo Fisher Scientific). Cell lysates were fractionated in 10% SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (0.45-μm IPVH00010; Merck) for 1 h at 100 V (fixed) at 10°C using a Mini Trans-Blot transfer system (Bio-Rad Laboratories, Hercules, CA, USA). Blotted proteins were visualized with Ponceau S (Merck, P17170) to examine the amounts of the transferred proteins. PVDF membranes were then incubated with primary and secondary antibodies (Supplementary Table S1). Results were investigated using a ChemiDoc XRS Plus instrument (Bio-Rad). Immunoprecipitation was conducted by protein A/G beads coated by indicated antibodies (Tanigawa et al. 2013 (link); Pan et al. 2010 (link)).

Co-IP procedures were carried out at 4 °C. HEK293T cells were seeded in two 10 cm dishes, at 2.5 × 106 cells per dish. 24 h later, cells were washed with ice-cold PBS, scraped in 2 mL IP buffer (50 mm HEPES, 250 mm NaCl, 5 mm EDTA, 0.1% Nonidet P-40, 2.5 mm sodium orthovanadate, 2.5 mm β-glycerophosphate) plus protease inhibitor cocktail (Roche Diagnostics) and sonicated. Cleared lysates were immunoprecipitated using anti-FUS (5 µg/mg lysate, A300-302A, Bethyl Laboratories, Inc.) overnight at 4°C. We used Dynabeads Protein G superparamagnetic beads from Life Technologies Inc. for IP with anti-FUS (5 µg/mg lysate, A300-302A, Bethyl Laboratories, Inc.). Co-IP proteins were then washed three times in IP buffer, resuspended in sample buffer, boiled, and subjected to 10% SDS–PAGE. Protein interaction was detected with immunoblotting using anti-MBLN1 (3A4, sc-47740, Santa Cruz).
Nuclear-cytosolic fractionation procedures were carried out at 4°C according to the manufacturer’s instructions (NE-PER 78833, Thermo Scientific). Samples were analyzed by SDS–PAGE as described above.