PP2A Activity Assay

Cells (1 × 10⁶ cells) were treated with siRNA (20 nM) or FTY720 (5 μM) for 4 hours and then lysed using NP-40 lysis buffer. PP2A activity was measured using a PP2A Immunoprecipitation Phosphatase Assay Kit (17–313, EMD Millipore). Briefly, protein lysates were incubated with PP2A antibody at 4°C with continuous rotation for 2 hours. Following the addition of assay buffers and malachite green solution, the plate was read at an absorbance of 650 nm using a microplate reader (Epoch Microplate Spectrophotometer). Phosphatase activity was determined using a standard curve. Experiments were repeated at least in triplicate, and phosphatase activity was reported as mean fold change ± SEM from the untreated sample for each cell line.