Triplicate protein samples of day 56 differentiated neurons in indirect contact co-culture mode (n = 2) were collected from Line B. The cells were solubilized in 22 μL of Laemmli sample buffer. After heating at 95°C for 5 minutes, samples were loaded on a 10% SDS gel (mini-Protean 3 from Bio-Rad) and run for 45 minutes at 100 V. The gel was fixed overnight in 50% ethanol/3% phosphoric acid, stained in colloidal coomassie blue for 30 minutes, washed three times for 10 minutes in water, and each sample lane cut into two fractions and chopped into about 1 mm by 1 mm pieces. After destaining in 50 mM NH3 HCO3−50% acetonitrile/50 mM NH3HCO3 the gel pieces were dried in Speedvac, the tryptic peptides were analyzed using Triple TOF 5600+ MS (Sciex) coupled with an Ultimate 3000 LC system (Dionex, Thermo Scientific). Peptides were trapped on a 5 mm Pepmap 100 C18 column (300 μm i.d., 5μm particle size, Dionex) and fractionated on a 200 mm Alltima C18 column (300 μm i.d., 3 μm). Acetonitrile concentration in the mobile phase in
