Assays

For diGly proteomics: HeLa cells were cultured in lysine- and arginine-free DMEM supplemented with dialyzed FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin and light (K0) lysine (38 μg/mL) and arginine (66 μg/ml). Medium and heavy media were the same except the light lysine was replaced with K4 and K8-lysine, respectively. Medium and heavy labeled cells were treated for 1 h with 250 µM LLOMe while light labeled were treated for 1 h with vehicle alone (EtOH). Light and heavy ...

HeLa cells stably expressing CNN2-APEX2 were grown at 37°C in DMEM supplemented with FBS, L-Glutamine, Sodium pyruvate. Cells were differentially treated with 5 µM NMS-873 for 15 min followed by 1 h 1 mM LLOMe (Sigma) and 2 h washout wihout any drugs. Proximity labeling was performed essentially as described before (Korver et al., 2019). Briefly, cells were incubated with 500 µM Biotin-Phenol during the last 30 min and subsequently pulsed by addition of H2O2 for 1 min at room temperature. To stop ...

Immunoprecipitation: Cells grown in 2-4x15 cm cell culture plates per sample were harvested by scraping on ice and stored at -80. Lysis was performed for 30 min at 4°C with MCLB buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.5% NP40, 1x PhosStop,1x protease inhibitor) or Glycerol buffer (20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 0.5% TritonX, 1x PhosStop, inhibitor, 1x protease inhibitor). Samples were cleared from debris by centrifugation (20.000 g for 10 min at 4°C) and Ultrafree®-CL ...

LCL cells were cultured in lysine- and arginine-free DMEM (GIBCO) supplemented with 2 mM glutamine, 10% dialyzed FBS and antibiotics as well as with 146 mg/ml light (K0, Sigma) or heavy lysine (K8, Cambridge Isotope Laboratories) and 84 mg/ml light (R0, Sigma) or heavy (R10, CambridgeIsotope Laboratories) arginine, respectively. Cell pellets were lysed in 8 M Urea in 50 mM Tris-HCl pH 8.0. Protein concentrations were determined by a BCA assay and protein amounts were adjusted to equal concentrations. ...

Pellets of membrane-protected material were lysed with RIPA buffer containing quenchers (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1x cOmplete Protease Inhibitor Cocktail (Roche), 1x PhosSTOP 956 (Roche), 10 mM sodium ascorbate, 1 mM Trolox and 1 mM sodium azide), sonicated and centrifuged at 10,000x g for 10 min. The supernatant was incubated with Streptavidin-agarose (Sigma-Aldrich) overnight, which was balanced with RIPA buffer containing quenchers. After ...

Following HA-immunoprecipitation of C9orf72 from SMCR8 KO cells, proteins were precipitated with 20% TCA and incubated for 20 min on ice. After centrifugation at 20,000 x g, 4°C for 30 min, the supernatant was discarded, 10% cold TCA was added to pellets and centrifuged again. Pellets were washed 3x in cold acetone, centrifuged and then dried in a speed vacuum concentrator. For in-solution tryptic digestion, pellets were resolved in 50 mM ammonium bicarbonate (ABC) pH 8.0 with 10% acetonitrile ...

BV2 cells were cultured in full medium in 10 cm cell culture dishes until they were confluent. MLN4924 (500 nM), CSN5i-3 (1 μM), or solvent control (0.01% DMSO) were added and the culturing continued for 6 h. After the incubation, cells were washed once with PBS, removed from the cell culture plate with a scraper, collected in tubes, and centrifuged in 1.5 ml Eppendorf tubes for 3 min to remove remaining PBS buffer, snap-frozen, and stored at -80 °C until further processing. Thereafter cells were ...