Rabbit anti raly

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.

For RIP of endogenous RNP complexes, NSC-34 were UV-cross-linked, lysed in 10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl₂, 0.5% NP40, 1 mM dithiothreitol (DTT), protease, and RNase inhibitors for 3 h at –80°C and centrifuged at 10,000 × g for 20 min at 4°C. The protein concentration was measured by Bradford assay to use in the IP the same amount of sample. The supernatants were precleared and incubated overnight at 4°C with protein A magnetic beads (Invitrogen) coated with 2 μg of rabbit anti-RALY (Bethyl Laboratories), rabbit anti-FUS/TLS (Abcam) antibody, or normal rabbit IgG (Millipore). The beads were then washed four times with NT2 buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl_2, 0.05% NP-40, 1% Urea), and then RNA was isolated with TRIZOL (Life Technologies) and processed for qRT-PCR analysis.

NSC-34 or HeLa cells were lysed in CHAPS buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% CHAPS, 10% glycerol, protease inhibitor cocktail, phosphatase inhibitor cocktail) and centrifuged at 14,000 × g for 10 min at 4°C. For RNase treatment, cell extracts were treated with RNaseA (100 μg/ml) for 15 min. For IP, protein extracts were incubated in a total volume of 1 ml at 4°C with either protein A for rabbit antibodies or protein G for mouse antibodies magnetic beads coated with 2 μg of primary antibody: rabbit anti-HA (A190-108A; Bethyl Laboratories), rabbit anti-RALY (A302-069; Bethyl Laboratories), and rabbit anti-FUS/TLS (ab23439, Abcam). For RALY-MYC/DDK and FUS-HA coIP, 30 μl of anti-FLAG M2 Affinity Gel (Sigma-Aldrich) were used. Beads were washed four times with CHAPS buffer, solubilized in reducing (DTT-containing) sample buffer, and analyzed by SDS–PAGE and Western blotting. After immunoblotting with primary antibodies, Western blot membranes were incubated with either mouse anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (211-032-171, Jackson Laboratories) or Clan-Blot IP detection kit HRP (21232, Thermoscientific) to minimize IgG heavy and light-chain signal interference.