Assays

For proteomic analysis, iPSC-derived neural stem cells at day 15 of culture were treated with 3.3 uM Nocodazole (Sigma) or same volume of DMSO (control) before being washed with ice-cold PBS, scrapped and centrifuged for 10 min at 300 g before lysis in Buffer A, containing 50 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100, 0.1% SDS, protease inhibitor (Pierce), at a volume twice that of the pellet. For neurons, day 40 of cultures treated only with DMSO were used. Samples ...

Library-Matched Single Shot (LMSS) method Mice were sacrificed by cervical dislocation and brains were subsequently extracted and put into cold phosphate buffered saline (PBS). The ventricular walls were laid bare by removing the dorsal ventricular wall and all tissue above it, as well as the choroid plexus. Brains were then snap-frozen on dry ice and cut into 100 μm sections on a cryostat (Leica CM1000S). The medial (MEZ) and lateral ventricular (SEZ) walls were then manually dissected under a ...

Mice were sacrificed by cervical dislocation (n=4) and brains were subsequently extracted and the injury-site and corresponding area on the contralateral side was removed using a 2.5 mm biopsy punch and the white matter was removed. Samples were homogenized using a (100 μl) dounce (Wheaton #357844) in 100 ul PBS (with protease inhibitor cocktail and Ethylenediaminetetraacetic acid (EDTA)) and directly frozen in liquid nitrogen and stored in -80 °C until tissue protein fractionation. Following ...

Nuclear extracts of cells expressing Trnp1, FLAGTrnp1, delta1-16Trnp1 or FLAGdelta1-16Trnp1 were obtained by re-suspended cells for 30 min on ice in Tween20 lysis buffer (25 mM HEPES pH 8, 20 mM NaCl, 2 mM EDTA, 1 mM PMSF, 0.5% Tween20, 1X protease inhibitors, cOmplete, Roche), pelleted nuclei by centrifugation, discarding the cytoplasmic fraction (supernatant) and lysing nuclei by sonication with lysis buffer containing 500 mM NaCl. DNAseI (250U; SIGMA) was added to the nuclear pellet and incubated ...

Per sample 10μg of isolated mitochondria were used. SDS was added to a final concentration of 2% for efficient solubilization, prior to tryptic protein digest using a modified FASP protocol (Wiśniewski et al., 2009). Proteomic measurements were performed on a Q-Exactive HF mass spectrometer (Thermo Scientific) online coupled to an Ultimate 3000 nano-RSLC (Dionex). Peptides were separated on a C18 nanoEase MZ HSS T3 column (100Å, 1.8 µm, 75 µm x 250 mm; Waters) in a 95 min non-linear acetonitrile ...

8 months old control C57BL/6J (n=10), 17 months aged C57BL/6J (n=4), and 8 months old APP/PS1 mice (n=5) were sacrificed through cervical dislocation, brains were removed and placed into cold 1x PBS. Biopsy punches of the visual cortex of both hemispheres were taken with a tissue puncher (2.5 mm diameter) and meninges and white matter were carefully removed using forceps to have a tissue sample consisting of grey matter only. Each sample was put into a low-protein binding Eppendorf tube, frozen ...

3-4 months old male and female C57BL/6J mice (n=10 intact controls, n=10 SW-injured, n=5 LPS injected) were sacrificed through cervical dislocation, brains were removed and placed into cold PBS. Biopsy punches (2.5 mm diameter) of the visual cortex of both hemispheres were dissected whereas meninges and white matter were carefully taken off. The contralateral (uninjured) cortices of the SW-injured brains were not considered for proteome analysis (intact: n=20, SW: n=10, LPS: n=10). Samples were ...