Assays

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 ...

Total protein content of CSF samples was measured by Bradford assay (BioRad, Feldkirchen, Germany)and 10μg per sample were proteolysed by the commercially available in-StageTip-NHS kit (PreOmics GmbH, Martinsried, Germany) according to the manufacturer's protocol. Briefly, CSF was reduced and alkylated and incubated for 3 hrs at 37°C with Lys-C and trypsin. Resulting peptides were dried for short term storage at -80°C. Prior to measurement, peptides were resuspended in 2% acetonitrile and 0.5% ...

Proximity-proteomics-based autophagosome content profiling to identify a role for LC3C in maintaining basal mitochondrial homeostasis. Selected mitochondrial proteins, including MTX1, were targeted by LC3C and p62 through a piecemeal mitophagy pathway. SILAC cells biotinylated using APEX proximity labeling. Cell lysates treated with protease. RIPA soluble and insoluble fractions were subjected to Streptavidin pulldown followed by in gel digestion. 4 lanes were cut from one sample.