Assay type 'Experimental Assay Type'

Corpus callosum dissections were lysed in 300 µL STET lysis buffer (1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, 50 mM TrisHCl pH 7.5) with a Precellys Evolution homogenizer (Bertin, Germany) using 0.5 mL soft tissue homogenization kit CK14 applying two cycles of 30 s with a speed of 6500rpm. After 15 min incubation on ice, samples were centrifuged at 16,000×g for 15 min to remove undissolved material and cell debris. The supernatant was transferred to a fresh protein lobind tube (Eppendorf, ...

Secretome analysis of primary neuronal cultures was performed using the high-performance secretome protein enrichment with click sugars" (hiSPECS) method, described in detail previously (Tüshaus et al, 2020). In brief, neurons were cultured for 48 h (DIV 5-7) in the presence of 50 µM ManNAz (#88904, ThermoFisher), cultivation media was filtered through 0.45 µm spin columns (Sigma-Aldrich, CLS8163). Glycoproteins were enriched using ConA agarose beads (Sigma, C7555) and clicked to magnetic DBCO ...

Samples were prepared by in solution digestions. For details, see the methods part of the accompanying paper.

Blood samples (20 ml) from clinically affected homozygous Npc1 mutation carriers and healthy donors were collected. Negative selection of peripheral blood monocyte-derived macrophages was performed by incubating full blood for 20 min at RT with RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies). An equal volume of washing buffer (D-PBS/2% FBS/1 mM EDTA) was added to each sample and layer of macrophages was separated from red blood cells and plasma by centrifugation on a Ficoll ...

After washing the primary cells with 1x PBS, cell-type specific growth media containing serum supplements with 50 µM of ManNAz (Thermo) was added for 48h. Afterwards, conditioned media was collected and filtered through Spin-X 0.45 µM cellulose acetate centrifuge tube filter (#8163, Costar) and stored at -20°C in protein Lobind tubes until further usage. Glycoprotein enrichment was performed using 60 µL Concanavalin A (ConA) bead slurry per sample (Sigma). ConA beads were washed twice with 1 mL ...

LUHMES cell culture LUHMES cells were cultured and differentiated as described previously (Sholz et al., 2011). 0.5 Million undifferentiated LUHMES (unLUHMES) cells were seeded into a Poly-D-Lysine coated 6-well containing growth media (DMEM F12, 1% N2 Supplement, 0.04 µg/mL bFGF) and harvested at day three in vitro. The live cell count was around 1 Million cells using Trypan blue and an automated cell counter (Biorad). For differentiation, 1 Million unLUHMES were seeded into a Poly-D-Lysine ...

The neurons were lysed direct on the plate in RIPA lysis buffer using a cell scraper. The lysate was transferred into a fresh Eppendorf tube and undissolved material was removed by centrifugation for 5 min at 20,000 g and 4°C. A protein assay was performed and 15 µg of protein were subjected to proteolytic digestion with the SP3 protocol. Proteins were reduced by addition of 9 µL of 200 mM dithiothreitol (Biozol, Germany) in 50 mM ammonium bicarbonate and incubation for 30 min at 37°C. Cysteine ...

The exosome pellets were lysed in 80 µL of a modified RIPA lysis buffer (50 mM TrisHCl pH 8, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v)) with protease inhibitors (Sigma Aldrich, US) on ice with intermediate vortexing. 20 µL H2O, 10 µL 100 mM MgCl2, and 25 units Benzonase (Sigma Aldrich, US) were added followed by an incubation for 30 min at 37°C at 1400 rpm in a Thermomixer (Eppendorf, Germany). Undissolved material was removed by centrifugation for ...

Primary microglia were isolated from mouse brains (cerebrum) using MACS Technology (Miltenyi Biotec) according to manufacturer´s instructions and as previously described (Daria et al., 2017). Briefly, brain cerebrum was dissected, freed from meninges and dissociated by enzymatic digestion using a Neural Tissue Dissociation Kit P (Miltenyi Biotec) and subsequent mechanical dissociation using 3 fire-polished glass Pasteur pipettes of decreasing diameter. CD11b positive microglia were magnetically ...

2 x 108 wild-type or Tspan15-knockout HEK-293T cells were lysed in 1% digitonin lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich). Lysates were pre-cleared with protein G sepharose prior to immunoprecipitation with Tspan15 mAb 1C12 chemically cross-linked to protein G sepharose with dimethyl pimelimidate (Thermo Fisher Scientific). Five independent immunoprecipitations were carried out for each cell type. Immunoprecipitation samples in non-reducing Laemmli buffer were subjected ...

Microglia isolated by MACS from WT and Myd88-/- mouse pups were seeded at a density of 1×106 cells per 60-mm dish in DMEM/FCS/L929 medium. After 2 DIV, microglia were washed with warm DMEM/pyruvate medium and treated with 20 µg/mL myelin debris (or HEPES control) in 2 mL of TIC medium for 4 h. After treatment, the cells were washed with DMEM/pyruvate medium, and incubated with 4 mL of DMEM containing 0.2% BSA for 16 h. The cells in each dish were washed with 2 mL of cold PBS on ice, and lysed ...

Samples for mass spectrometry were obtained from 8- to 9-week-old, male mice. Mitochondria were immunopurified from cerebellum according to the described protocol with the alteration that the final mitochondrial pellet was washed twice in IB without EDTA and BSA. Sample were lysed in 100 µL SDT lysis buffer (4% w:v SDS, 100 mM DTT in 100 mM Tris-HCl pH 7.6) by heating for 5 min at 95°C and ultrasonication (Vialtweeter: 6 times for 30 s, 100% Amplitude, 50% cycle, max power; Hielscher Ultrasonics). ...

Nine patients per group were treated with either acitretin or vehicle control. Cerebrospinal fluid was collected before (baseline value) and after treatment. A volume of 5 µL of CSF per sample was subjected to proteolytic digestion in 50 mM ammonium bicarbonate with 0.1% sodium deoxycholate (Sigma Aldrich, Germany) as previously described (Pigoni et al., 2016). Briefly, protein disulfide bonds were reduced with dithiothreitol and sulfhydryl residues were alkylated using iodoacetamide. Proteins ...

Cells were harvested and centrifuged 400g for 10 min at 4°C. Cell pellets were resuspended in STE-Buffer (250mM sucrose, 5mM Tris pH 7, 1mM EGTA, PI mix 1:500) and lysed with a 27-gauge needle. Samples were centrifuged 10 min at 800g to remove nuclei, then 10 min at 15.000g to remove mitochondria and finally 1 hour 100.000g. The resulting pellets were washed twice with 100mM Na2CO3 and centrifuged 30 min at 100000g after each wash. Pellets from membrane preparations were dissolved in lysis buffer ...

Post-nuclear supernatants were prepared from testis of wild type and SPPL2c-/- mice (n=5 per genotype). After removal of the tunica albuginea, testicles from two wild type mice were minced with an ultraturrax in 250 mM sucrose, 10 mM HEPES-NaOH, pH 7.4 and 1 mM EDTA (HB, homogenisation buffer) and then further homogenised by eight strokes of a Potter homogeniser. For sedimentation of nuclei, the tissue homogenate was centrifuged at 750 x g for 10 min. The supernatants were collected as post-nuclear ...

human IPSCs from control or patients with NDUFS4 mutations were differentiated into proliferating and non-proliferating astrocytes. Three control and 3 patients lines were analyzed. Per each line, 3 biological replicates were collected at 3 stages: IPSC, proliferating astrocyte and non-proliferating astrocytes.

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

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

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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. ...

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 ...

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 ...

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 ...

Sample preparation for LC-MS/MS Eight million of Cy3/5-positive autophagosomes (roughly 10ug of proteins) were denatured with 2% sodium deoxycholate, 50 mM Tris-HCl pH 8.5, 2.5 mM TCEP, 10 mM chloroacetamide at 95°C for 10 min. Lysates were prepared with in-StageTip (iST) processing method for LC-MS/MS as previously described by Kulak et al. 2014. Briefly, Proteins were digested overnight at 37°C with 1 volume of 50mM Tris-HCl pH 8.5 containing LysC (Wako Chemicals) at 1:100 (w/w) ratio and Trypsin ...

Samples were essentially prepared as described in Zellner et al. Mol Cell 2021 (DOI: 10.1016/j.molcel.2021.01.009)

APEX2-mediated biotinylation of cells was carried out as described before (Hung et al., 2016). In brief, 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. All steps were carried out at 4°C unless stated otherwise. Cells were washed and suspended in homogenization ...

Cells were incubated with 500 µM Biotin-Phenol during the last 30 min of LLOMe or GPN treatment and subsequently pulsed by addition of H2O2 for 1 min at room temperature. To stop the biotinylation reaction, they were washed 3x with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox in DPBS) and 3x with PBS. All further steps were performed at 4°C unless indicated otherwise. After cell harvest with 0.25% Trypsin/EDTA (ThermoFisher Scientific), cells were counted and heavy- ...

Frozen cell pellets from 4x15 cm cell culture plates were lysed in Glycerol buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.5 % Triton-X-100, 10 % Glycerol, 1x protease inhibitor, 1x phosphatase inhibitor) for 30 min at 4° C with end-over-end rotation. Lysates were cleared from cell debris by centrifugation prior to adjustment of protein concentrations between the samples and overnight immunoprecipitation at 4° C with pre-equilibrated anti-HA-agarose (Sigma). Agarose beads were washed five ...

HeLa cells stably expressing UBE2QL1-APEX2 were grown in lysine- and arginine-free DMEM supplemented with FBS, L-Glutamine, Sodium pyruvate, heavy arginine (R10) (38 μg/ml) and lysine (K8) (66 μg/ml) or light arginine (R0) (38 μg/ml) and lysine (K0) (66 μg/ml), respectively. Further experiments were conducted as soon as the cells reached a protein labelling with heavy amino acids of at least 95%. Heavy-labeled cells were treated with 250 μM Leu-Leu methyl ester hydrobromide (LLOMe, Sigma) for 3 ...

Cells (4x 15 cm dishes) were harvested and lysed with 3 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P40 (NP40) and EDTA-free protease inhibitor cocktail tablets). Centrifugation-cleared lysates (13,000 rpm) were filtered through 0.45 µm spin filters (Millipore Ultrafree-CL) and immunoprecipitated with 60 µl anti-HA resin. Resin containing immune complexes were washed five times with lysis buffer followed by five PBS washes, and elution with 150 µl of 250 mg/ml HA peptide in ...

Anti-HA-immunoprecipitation was performed as previously described (Behrends et al, 2010; Jung et al, 2015; Jung et al, 2017; Sowa et al, 2009). Summarily, expression of TAPL-HA and coreTAPL-HA was induced by addition of 4 µg/ml doxycycline for 24 h in HeLa Flp-In T-REx cells. Parental non-transfected HeLa Flp-In T-REx cells were used as negative control. For each sample, 6.4 x 107 cells were harvested, frozen in liquid nitrogen and stored at -80 °C. Cells were lysed in 3 ml MCLB buffer (50 mM ...

Samples were processed and analyszed as described in Behrends et al., Nature 2010.