HeLa cells (70% of confluence) were transfected with 3 μg of pFLAG-TDP-43 wild-type using the Effectene reagent. After 24 h, cell culture medium was removed and cells were washed with cold PBS and harvested. Cells were lysed in 500 μl of IP buffer (20 mM Tris pH 7.5, 110 mM NaCl, 0.5% Triton-X, 1× Complete Protease Inhibitor Cocktail) by sonication (3 min, mid power), in ice-cooled sonicating bath (BioRuptor, Diagenode, Belgium). The cell lysate was pre-cleared by incubation with 30 μl Protein A/G PLUS agarose beads (Santa Cruz Biotechnology Inc., Dallas, Texas, USA) in IP buffer for 1.5 h at 4°C. The pellets were discarded and the supernatants were used for immunoprecipitation: the cell lysates were incubated with 2 μg of mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) on a rotating device for an hour at 4°C. Then, 30 μl of Protein A/G PLUS agarose beads were added to each sample and incubated overnight at 4°C. The pellet was then washed three times in ice-cold IP buffer. The supernatants was discarded, and the pellet was re-suspended in 30 μl of 3× sample loading dye. The samples were fractionated by SDS-PAGE (10%) and analyzed by immunoblotting 1:2000 rabbit polyclonal anti-TDP-43 antibody (ProteinTech), with 1:500 rabbit polyclonal anti-DAZAP1 antibody and 1:500 rabbit polyclonal anti-hnRNP H antibody previously described (39 (link),40 (link)).