Assays

Proteomics: brain tissues coming from both human PFC and the 4 different mouse models were prepared as follows. Tissues were grinded with a biomasher using 350 µL of MeOH:H2O (4:1). Protein pellets were resuspended in 200 µL Laemmli buffer (10% SDS, Tris 1M pH 6.8, glycerol) then centrifuged at 11.135 rpm at 4°C for 5 minutes. Protein concentration was determined using DC assay (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. 100 µg of protein lysate for each sample were ...

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 ...

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 ...