Phosphatase inhibitor 3

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).