Assays

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...