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 ...

3-4 months old male and female C57BL/6J mice (n=10 intact controls, n=10 SW-injured, n=5 LPS injected) were sacrificed through cervical dislocation, brains were removed and placed into cold PBS. Biopsy punches (2.5 mm diameter) of the visual cortex of both hemispheres were dissected whereas meninges and white matter were carefully taken off. The contralateral (uninjured) cortices of the SW-injured brains were not considered for proteome analysis (intact: n=20, SW: n=10, LPS: n=10). Samples were ...

Total protein content of CSF samples was measured by Bradford assay (BioRad, Feldkirchen, Germany)and 10μg per sample were proteolysed by the commercially available in-StageTip-NHS kit (PreOmics GmbH, Martinsried, Germany) according to the manufacturer's protocol. Briefly, CSF was reduced and alkylated and incubated for 3 hrs at 37°C with Lys-C and trypsin. Resulting peptides were dried for short term storage at -80°C. Prior to measurement, peptides were resuspended in 2% acetonitrile and 0.5% ...

Proximity-proteomics-based autophagosome content profiling to identify a role for LC3C in maintaining basal mitochondrial homeostasis. Selected mitochondrial proteins, including MTX1, were targeted by LC3C and p62 through a piecemeal mitophagy pathway. SILAC cells biotinylated using APEX proximity labeling. Cell lysates treated with protease. RIPA soluble and insoluble fractions were subjected to Streptavidin pulldown followed by in gel digestion. 4 lanes were cut from one sample.

Samples were processed and analyszed as described in Behrends et al., Nature 2010.

Anti-HA-immunoprecipitation was performed as previously described (Behrends et al, 2010; Jung et al, 2015; Jung et al, 2017; Sowa et al, 2009). Summarily, expression of TAPL-HA and coreTAPL-HA was induced by addition of 4 µg/ml doxycycline for 24 h in HeLa Flp-In T-REx cells. Parental non-transfected HeLa Flp-In T-REx cells were used as negative control. For each sample, 6.4 x 107 cells were harvested, frozen in liquid nitrogen and stored at -80 °C. Cells were lysed in 3 ml MCLB buffer (50 mM ...

Cells (4x 15 cm dishes) were harvested and lysed with 3 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P40 (NP40) and EDTA-free protease inhibitor cocktail tablets). Centrifugation-cleared lysates (13,000 rpm) were filtered through 0.45 µm spin filters (Millipore Ultrafree-CL) and immunoprecipitated with 60 µl anti-HA resin. Resin containing immune complexes were washed five times with lysis buffer followed by five PBS washes, and elution with 150 µl of 250 mg/ml HA peptide in ...