2.6. PP2A Activity Assay

The PP2A activity was measured according to the manufacturer's protocol. The cells were harvested and homogenized in 1 ml of lysis activity buffer included (20 mM imidazole-HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0, with 10 μg/ml each of aprotinin, leupeptin, 1 mM benzamidine, and 1 mM PMSF). The cells were collected in Eppendorf tubes, homogenized for 10 minutes at 4°C, and centrifuged at 11000 × g for 15 minutes at 4°C. The protein concentration was measured using Bradford protein assay method. 200 μg of total protein of each sample was transferred to new Eppendorf tubes, including 30 μl of protein A agarose and 4 μl of normal mouse IgG, and the volumes were topped off to 1000 μl with pNPP assay buffer. The NIgG immunoprecipitates were incubated for one and half hour at 4°C with constant rocking and followed by centrifugation at 3000 × g for 3 minutes at 4°C. The supernatants were transferred to new Eppendorf tubes, including 30 μl of protein A agarose and 4 μl of anti-PP2Ac antibody, and the volumes were topped off to 1000 μl with pNPP assay buffer. All tubes were incubated at constant rotated for 2 hours at 4°C and centrifuged at 3000 × g for 3 minutes at 4°C. The immunoprecipitated PP2Ac and NIgG beads were washed 3 times with 700 μl Tris-buffered saline (TBS) (3000 × g for 3 minutes at 4°C), once with 500 μl pNPP Ser/Thr assay buffer, followed by incubation with 60 μl of 750 μM of threonine phosphopeptide (K-R-pT-I-R-R) and 20 μl of pNPP Ser/Thr assay buffer at 30°C for 10 minutes in a shaking incubator. The beads were centrifuged 3000 × g for 3 minutes at 4°C, and 25 μl of the supernatants was mixed with 100 μl of Malachite green phosphate detection solution into each well to a 96-well plate microtiter plate to terminate the reaction, and the color was allowed to develop for 15 minutes at room temperature. The absorbance of the samples was read using a microplate reader at 650 nm. The amount of free phosphate released was calculated from a standard curve (according to the manufacturer's protocol) and normalized to that of the controls.