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 ...

Dissection of ventral pial cerebral vessels: Mice were anesthetized with Ketamine (100 mg/kg) and Xylazine (10 mg/kg) and transcardially perfused with 20 ml PBS, followed by 2 ml of 2% Evan’s blue (w/v) prepared in PBS. After brain harvest, pial vessels were collected from the ventral face of the brain with micro forceps under a M205 A dissection microscope (Leica) and snap frozen on dry ice. Lysis of cerebral vessels. Vessels were mixed with 4% (w/v) SDS, 100 mM dithiothreitol, 100 mM Tris–HCl, ...

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.