Assays

Phosphoproteomics: starting from the protein extracts from the global proteomics experiments, proteases inhibitors (Sigma, P8340) and phosphatases inhibitors (final concentration in Na3VO4 = 1 mM) were added to all samples. Protein concentration was determined using RC-DC assay (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. 250 µg of proteins for each sample were reduced and alkylated prior to an in-house optimized single-pot, solid-phase-enhanced sample preparation ...

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 ...

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 ...

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, ...

Cells were collected and lysed in urea buffer (9 M Urea, 50 mM Tris pH 8, 150 mM NaCl, 1x Roche protease inhibitor cocktail) followed by short sonification. Samples were cleared by centrifugation and protein amounts were adapted. Protein reduction was performed with dithiothreitol (DTT; 5 mM final) for 25 min at 56°C and protein alkylation by the addition of iodoacetamide (14 mM final) for 30 min at room temperature. Protein mixtures were quenched with DTT and diluted 1:5 with 1 M Tris-Hcl, pH ...