Assays

ollowing 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 ...

All samples were reconstituted in 0.1% formic acid and separated using an Easy-nLC1200 liquid chromatograph (Thermo Scientific) followed by peptide detection on a Q Exactive HF mass spectrometer (Thermo Scientific). Samples were separated on a 75 µm x 15 cm custom-made fused silica capillary packed with C18AQ resin (Reprosil-PUR 120, 1.9 µm, Dr. Maisch), flow rates and gradients were adjusted according to the experiment. Except for plasma membranome analysis, peptide mixtures were separated on a ...

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 ...

Proximity labeling: Cells were supplemented with 500 µM biotin-phenol (IrisBiotech) for 30 min at 37°C before addition of 1 mM H2O2 at room temperature. Cells were then first washed with quencher solution (1 mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox in DPBS), then with DPBS, scraped and harvested. Proteinase K digest: All steps were carried out at 4°C unless stated otherwise. Cells were washed and suspended in homogenization buffer I (10 mM KCl, 1.5 mM MgCl2, 10 mM HEPES-KOH and 1 ...

Samples were lysed with lysis buffer (2% SDS, 50mM Tris-HCl pH 8.5, 10mM TCEP, 40mM chloroacetamide and protease inhibitor cocktail tablet [EDTA-free, Roche]). Samples were incubated for 5 minutes at 95°C before sonication with Sonic Vibra Cell at 1s ON/ 1s OFF pulse for 30s at a maximal amplitude of 30% to shear genomic DNA. After sonication, samples were incubated for 10min at 95°C. Proteins were precipitated using 3 volumes of ice-cold methanol, 1 volume Chloroform and 2.5 volumes ddH2O. After ...

pAstros were transduced with control or Ngn2-expressing retrovirus. 2DPT, media was replaced with differentiation media in absence of presence of AMG; 20DPT, DsRed-positive cells were sorted, barcoded with CMOs and analyzed by scRNAseq via 10XGenoics platform

pAstros were transduced with control or Ngn2-expressing retrovirus. 2DPT, media was replaced with differentiation media in absence of presence of AMG; 5DPT, DsRed-positive cells were sorted, barcoded with CMOs and analyzed by scRNAseq via 10XGenoics platform