Phosphatase inhibitor set 2

HD and C116 NSC were cultured as described above. Cells were plated at a density of 100,000 per cm² on Matrigel (Corning, CB40234)-treated six-well plates. Cells were transfected at about 90% confluency. Before transfection, the medium was changed to antibiotic-free medium. siRNAs (Dharmacon, FKBP5: J-004224009, non-targeting 001206–13-20) were diluted into a stock solution of 10,000 nM, and 3 μL was used per well in a six-well plate (25 nM per well). The siRNA was complexed with 6 μL of siLentFect Lipid reagent (Biorad, 170–3361). The siRNA and siLentFect were incubated for 20 min at room teMPERature (RT) and then slowly added dropwise to the well, and the plate was gently swirled. Fresh antibiotic-free medium was applied 48 h after transfection, and the cells were harvested 96 h after transfection on ice. Cells were rinsed twice with DPBS (Corning, 21–031-CV) and then collected by scrapping in MPER (Thermo Fisher Scientific, 78,501) with Complete Mini protease inhibitor cocktail (MilliporeSigma, 11,836,170,001), 1% Phosphatase Inhibitor Set II (EMD Millipore, 524,625), and 1% Phosphatase Inhibitor III (EMD Millipore, 539,134). Protein concentrations were determined using the BCA assay (Thermo Fisher Scientific, 23,252).

NSC were derived and cultured as described [22 (link)]. After harvesting from rosettes, NSC were grown on Matrigel (Corning, CB40,234)-coated dishes in Neural Proliferation Medium (Life Technologies,21,103–049). Neurobasal medium was supplemented with 1XB27 (Life Technologies, 17,504–044), 2 mM L-glutamine (Gibco, 35,050–061), 10 ng/mL leukemia inhibitory factor (Peprotech, 300–05), 25 ng/mL bFGF (Peprotech, 100–18B), and 1% penicillin-streptomycin (Corning, 15,140–122). NSC were passaged with Accutase (A11105-01) every 7 d, passaging at a 1:3 ratio. Lysates were harvested by cell scrapping in MPER (Thermo Fisher Scientific, 78,501) with Complete Mini protease inhibitor cocktail (MilliporeSigma, 11,836,170,001), 1% phosphatase Inhibitor Set II (EMD Millipore, 524,625), 1% phosphatase Inhibitor Set III (EMD Millipore, 539,134), 50 μM tricostatin A (MilliporeSigma, T8552), 30 μM sodium butyrate (MilliporeSigma, 303,410), and 30 mM nicotinamide (MilliporeSigma, 72,340). Lysate was sonicated once at 40 mA with 5 s pulses, 5X, with 5 s breaks between each pulse. Sonicated lysate was then centrifuged at 15,294 g for 20 min at 4°C. The supernatant was transfer to a new tube. Protein concentration of the supernatant was determined using the BCA assay (Thermo Fisher Scientific, 23,252).