Assays

8 months old control C57BL/6J (n=10), 17 months aged C57BL/6J (n=4), and 8 months old APP/PS1 mice (n=5) were sacrificed through cervical dislocation, brains were removed and placed into cold 1x PBS. Biopsy punches of the visual cortex of both hemispheres were taken with a tissue puncher (2.5 mm diameter) and meninges and white matter were carefully removed using forceps to have a tissue sample consisting of grey matter only. Each sample was put into a low-protein binding Eppendorf tube, frozen ...

Per sample 10μg of isolated mitochondria were used. SDS was added to a final concentration of 2% for efficient solubilization, prior to tryptic protein digest using a modified FASP protocol (Wiśniewski et al., 2009). Proteomic measurements were performed on a Q-Exactive HF mass spectrometer (Thermo Scientific) online coupled to an Ultimate 3000 nano-RSLC (Dionex). Peptides were separated on a C18 nanoEase MZ HSS T3 column (100Å, 1.8 µm, 75 µm x 250 mm; Waters) in a 95 min non-linear acetonitrile ...

Nuclear extracts of cells expressing Trnp1, FLAGTrnp1, delta1-16Trnp1 or FLAGdelta1-16Trnp1 were obtained by re-suspended cells for 30 min on ice in Tween20 lysis buffer (25 mM HEPES pH 8, 20 mM NaCl, 2 mM EDTA, 1 mM PMSF, 0.5% Tween20, 1X protease inhibitors, cOmplete, Roche), pelleted nuclei by centrifugation, discarding the cytoplasmic fraction (supernatant) and lysing nuclei by sonication with lysis buffer containing 500 mM NaCl. DNAseI (250U; SIGMA) was added to the nuclear pellet and incubated ...

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 ...