Wash buffer b

PLA was conducted according to manufacturer’s protocol (Sigma-Aldrich). Briefly, after fixation, permeabilization with 0.1% Triton X-100, and 40 min blocking, cells were incubated at room temperature for 2.5 h with primary antibodies: goat anti-FUS (1:300; Sigma-Aldrich) and rabbit anti-RALY (1:300; Bethyl). As negative controls, either no primary antibodies or goat anti-DYNACTIN1 (1:100; Novus Biologicals) and rabbit anti-RALY were incubated. After two 10-min washings with PBS, cells were incubated with Duolink In Situ PLA Probe anti-Rabbit PLUS and anti-Goat MINUS (Sigma-Aldrich), diluted 1:5 in blocking solution for 1 h at 37°C. After two 5-min washings in Wash Buffer A (Sigma-Aldrich), cells were incubated with ligase for 30 min at 37°C. After two 5-min washings in Wash Buffer A, cells were incubated with polymerase and amplification solution for 100 min at 37°C, washed twice for 10 min in Wash Buffer B (Sigma-Aldrich), once in 0.01% in Wash Buffer B, dried at room temperature in the dark, and mounted with Mounting Media with DAPI (Sigma-Aldrich). For MNs, after washings in Wash Buffer B, cells were incubated with mouse anti-SMI32 antibody (1:500, Abcam) and hence with Alexa Fluor 488–conjugated goat anti-mouse.

HeLa cells cultured on glass coverslips were fixed in 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque) for 15 min, washed with PBS, and permeabilized with 0.2% Triton X-100. The proximity ligation assay (PLA) was performed according to the Duolink PLA Fluorescence Protocol (Sigma-Aldrich). The cultures were incubated in Duolink Blocking Solution at 37°C for 1 h, and then the blocking solution was removed and replaced with primary antibody solution diluted in Duolink Antibody Diluent overnight at room temperature. The coverslips were washed twice with Wash buffer A (Sigma-Aldrich) for 5 min each, then Duolink In Situ PLA Probe Anti-Rabbit PLUS and Duolink In Situ PLA Probe Anti-Mouse MINUS probes (Sigma-Aldrich) diluted in Duolink Antibody Diluent were applied, and then the coverslips were incubated in a humidified chamber for 1 h at 37°C. After washing the coverslips with Wash buffer A, ligase mixed with Duolink Ligation Buffer (Sigma-Aldrich) was applied and incubated for 30 min at 37°C. Then, the coverslips were washed twice with Wash buffer A, followed by the addition of amplification solution and incubation for 100 min at 37°C. The coverslips were washed twice in wash buffer B (Sigma-Aldrich) for 10 min each and then mounted with Prolong Diamond antifade (Life Technologies). Imaging was performed using a Confocal Laser Scanning Microscope LSM710 (Carl Zeiss).