Assays

Parental and ITCH KO HeLa cells were cultured in lysine- and arginine-free DMEM supplemented with dialyzed FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin and light (K0) lysine (38 μg/mL) and arginine (66 μg/ml). Heavy medium was the same except the light lysine was replaced with K8-lysine (L-Lysine, 2HCl U-13C U-15N, Cambridge Isotope Laboratories Inc). All cells were treated for 1 h with 1mM LLOMe (Sigma). Subsequently, cells were processed as described before (Fiskin et ...

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, ...

Blood samples (20 ml) from clinically affected homozygous Npc1 mutation carriers and healthy donors were collected. Negative selection of peripheral blood monocyte-derived macrophages was performed by incubating full blood for 20 min at RT with RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies). An equal volume of washing buffer (D-PBS/2% FBS/1 mM EDTA) was added to each sample and layer of macrophages was separated from red blood cells and plasma by centrifugation on a Ficoll ...

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 ...