Assays

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 ...

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 ...

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 ...

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). ...

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 ...

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 ...

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 ...