Quantification of PP2A Phosphatase Activity

Hippocampal neurons were initially lysed in accordance with the instructions provided in the Serine/Threonine Phosphatase Assay Kit (V2460, Promega). Lysates were then centrifuged at 1 × 10⁵g at 4°C for 1 hour in phosphatase storage buffer [2 mM EGTA, 5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 150 mM NaCl, 1% Triton X-100, 50 mM tris-HCl (pH 7.4), and 0.5% protease inhibitor cocktail]. Sephadex G-25 spin columns were used to remove free phosphate found endogenously, followed by incubation for 1 hour at 37°C in PP2A reaction buffer [250 mM imidazole (pH 7.2), 1 mM EGTA, 0.1% β-mercaptoethanol, and BSA (0.5 mg/ml)] supplemented with Ser/Thr phosphopeptide. The reaction was stopped by adding 50 μl of molybdate dye/additive mixture. After 30 min, absorbance was measured at 600 nm in a 96-well microplate reader (Tecan). PP2A activity measurements were normalized to the total DNA content in biological replicates using a CyQUANT assay (Thermo Fisher Scientific). PP2B and PP2C show very low to no detectable activity in the presence of EGTA (PP2B) and EDTA (PP2C); it is also noteworthy that the phosphopeptide used in this assay is a poor substrate for protein phosphatase 1.