Sep pak c18 5cc vacuum cartridges

The cell pellets were incubated with 6 ml of preheated SDC buffer containing 1% sodium deoxycholate (SDC, Sigma–Aldrich), 40 mM 2-cloroacetamide (CAA, Sigma–Aldrich), 10 mM tris(2-carboxyethyl)phosphine (TCEP; Thermo Fisher Scientific) and 100 mM Tris, pH 8.0. After incubation for 2 min at 95 °C, the samples were ultrasonicated for 2 min with 0.5 seconds pulse (50% intensity) and 0.2 s pause (Sonopuls, Bandelin). Incubation and ultrasonication was repeated for a second time. After a final incubation for 2 min at 95 °C, 1/6 of the sample was diluted 1:2 with MS grade water (VWR). Proteins were digested overnight at 37 °C with 50 μg trypsin (Promega). The solution of peptides was then acidified with trifluoroacetic acid (Merck) to a final concentration of 1%, followed by desalting via Sep-Pak C18 5cc vacuum cartridges (Waters). The cartridge was washed twice with 1 ml of 100% methanol, twice with 1 ml of 0.1% FA in 80% ACN and twice with 1 ml of 0.1% FA in water prior to sample loading. After loading the acidified sample, the cartridge was washed twice with 1 ml of 0.1% FA in water. Elution was done with 2 × 1 ml of 0.1% FA in 80% ACN.

On DIV1, neurons were transduced with lentivirus-packaged myc-tagged SYNJ2BP WT plasmid. On DIV6, the cells were cultured in insulin-free NB + B27 + PSG for 2 h and treated with or without CC and insulin, respectively. The neurons were collected in ice-cold PBS and centrifuged for 1 min at 9,300g. The pellets were snap-frozen and stored at −80 °C. The cell pellets (~10 × 10⁶ mouse cortical neurons) were incubated with 400 µl preheated sodium deoxycholate buffer (1% sodium deoxycholate (Sigma-Aldrich), 40 mM 2-chloroacetamide (Sigma-Aldrich) and 10 mM tris(2-carboxyethyl)phosphine (Thermo Fisher Scientific) in 100 mM Tris, pH 8.0). Afterwards, the samples were boiled for 5 min at 95 °C and ultrasonicated for 10 min with 10 × 30 s at high intensity and a 30-s pause between each cycle (Bioruptor Plus sonication system, Diagenode). Incubation and ultrasonication was repeated. The samples were diluted 1:1 with MS grade water (VWR). Proteins were digested with 2 µg Lys-C (Wako) for 4 h and overnight at 37 °C with 4 µg trypsin (Promega). The solution of peptides was acidified with trifluoroacetic acid (TFA; Merck) to a final concentration of 1%, followed by desalting via Sep-Pak C18 5cc vacuum cartridges (Waters).