Data files

The microglial proteome of homozygous Cln3Δex7/8/Δex7/8 was compared with those of littermate control heterozygous (Cln3Δex7/8/+) or wildtype (Cln3+/+) mice at 3 (Cln3Δex7/8/Δex7/8 n=5 vs controls n=7) and 12 months (Cln3Δex7/8/Δex7/8 n=4 vs controls n=5) of age. Microglia were acutely isolated using MACS with CD11b microbeads (Miltenyi Biotec) as previously described in Colombo et al. 36 Sample preparation and mass spectrometric measurements using data independent acquisition were performed as ...

Wild-type (WT) and ADAM17 knockout (KO) mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), containing 1% L-Glutamine, 1% Penicilin and Streptomycin, 1% Sodium Pyruvate and 10% Fetal Bovine Serum (FBS) at 37 °C, 5% CO2 (all reagents were purchased from Euroclone, Pero, Italy). HTB94 were kindly provided by Prof Hideaki Nagase, and cultured in DMEM containing 1% L-Glutamine, 1% Penicillin and Streptomycin, 1% Sodium Pyruvate and 10% FBS at 37° C, 5% CO2. ...

HeLa WT and CTS DBLZ-deficient as well as SH-SY5Y WT and CTS DBL-deficient cell clones (each n=6) were seeded 24 h after cell pellet collection. Cells were washed in ice cold PBS and harvested with a cell scraper. A modified RIPA lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 50 mM TrisHCl pH 8) was used to lyse the cells one ice for 30 min. The supernatant was collected after centrifugation at 16.000 rpm for 10 min at 4°C. 10 μl were used to determine ...

Sample preparation for proteomics analysis was performed as described previously with slight modifications. Briefly, for mouse samples, SDC lysis buffer (2% SDC, 100 mM Tris-HCl pH 8.5) was used to lyse the cell pellets at 95°C for 45 min at 600 rpm in a thermoshaker. For human samples which were fixed in PFA, prior to the SDC lysis buffer step, the samples were first resuspended in 6% SDS buffer, heat denatured, sonicated and then precipitated using 80% acetone overnight in -20°C. Next day, these ...

After respective treatment, cells were washed 2 times with ice-cold DPBS, scraped in PBS and pellets either processed immediately or stored at -80°C. Cells were lysed in RIPA buffer for 30 min on ice. After clearance by centrifugation at 20,000 × g for 10 min at 4 °C, protein concentrations were adjusted using BCA assay. Cleared and adjusted supernatants were incubated overnight on an overhead rotator with preequilibrated streptavidin agarose (Sigma). Next day, beads were washed 2 times with RIPA ...

Tryptic in solution digestion of the purified myelin fraction was performed according to the filter-aided sample preparation (FASP) protocol (Erwig et al., 2019) originally described by Manza et al., 2005; followed by LC-MS-analysis. In brief, purified myelin fractions corresponding to 10 µg myelin protein were dissolved and lysed in lysis buffer (1% ASB-14, 7 M urea, 2 M thiourea. 10 mM DTT 0.1 M Tris pH 8.5). Homogenised samples were diluted with lysis buffer containing 2% CHAPS to reduce ASB-14 ...

Cerebrospinal fluid samples of patients with Parkinson’s disease and healthy controls were used in this study. After tryptic digestion, all samples were spiked with indexed retention time (iRT) peptides and were measured using a DIA mass spectrometry approach. The CSF samples were prepared for digestion using consecutively RapiGestTM SF Surfactant, DTT and iodoacetamide. Then, proteolytic digestion was performedby trypsin solution and stopped by TFA. Dried peptides were resuspended in TFA. The ...