Sigma protease inhibitor mix

HeLa cells (0.5–1×10⁶) were incubated with 10 mM TMR-DPRs (in medium lacking serum) for 2h and then harvested by scraping into PBS and centrifugation at 1,200 g for 5 min. The cell pellet was resuspended in 200 μl RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) supplemented with ~0.05U/μl benzonase (Sigma), 1x Sigma protease inhibitor mix and 1x phosphatase inhibitors (final concentration: 10 mM NaF, 1 mM β-glycerophosphate, 1 mM Na₃VO₄) and incubated on ice for 10–15 min. Cells were sonicated once for 45 s using a BioRuptorPico (Diagenode) and 10% of the lysate retained for the input. The rest of the lysate was centrifuged for 30 min at 13,000 g and 4°C to separate the RIPA-soluble proteins from the insoluble proteins. The RIPA insoluble pellet was resuspended and washed once in RIPA buffer, followed by 45 s sonication in the BioRuptorPico and subsequent centrifugation at 13,000 g for 30 min at 4°C. The RIPA insoluble pellet was finally lysed in 40 μl Urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-HCl, pH 8.5) and 45 s sonication using the BioRuptorPico. Samples were analyzed by anti-TDP-43 western blot. Note that the 4.5x overrepresentation of the pellet fraction for reasons of visibility in the western blot was corrected in the quantification.