PP2A activity assay

Cellular PP2A activity was assayed using a PP2A immunoprecipitation phosphatase assay kit (Upstate). Cells were washed in TBS, lysed with phosphatase assay buffer (20mM imidazole-HCl, 2mM EDTA, 2mM EGTA, 0.1% NP-40, pH 7.0, protease inhibitors). PP2A-C subunit was immunoprecipitated from total cell lysates (500 μg) using 4 μg of anti-PP2A-C antibody (clone 1D6, Upstate) and Protein A agarose for 2 h at 4°C. PP2A activity was assayed by incubating the immunoprecipitated protein with the synthetic phosphopeptide K-R-pT-I-R-R at 30°C for 10 min prior to detection with malachite green phosphate detection solution, according to the manufacturer's instructions. Phosphatase activity was normalized to the relative amount of immunoprecipitated C subunit using Western blot quantitation with Image J software.