Assays

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 ...

Comparative small RNA expression profiling analysis for four transgenic mouse models ( C9orf72, SOD1, TDP-43 and FUS) of ALS with each 5 males/females and 5 case/control samples. For FUS one male control sample was excluded.

Comparative gene expression profiling analysis of RNA-seq data for four transgenic mouse models ( C9orf72, SOD1, TDP-43 and FUS) of ALS with each 5 males/females and 5 case/control samples. For FUS one male control sample was excluded.

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.

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% ...

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 ...