Assays

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 ...

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 ...

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 ...