2.9. In vitro PP2A phosphatase assay

In vitro PP2A phosphatase activity was determined as described (Liu et al., 2010a), with some modifications. Briefly, after serum-starvation for 24 h, cells were incubated for 4 h in the absence or presence of resveratrol (0–100 μM) ± IGF-1 (10 ng/ml), with 6 replicates of each treatment. Subsequently, the cells were lysed in 50 mM Tris-HCl buffer, pH 7.0, containing 1% Nonidet P-40, 2 mM EDTA, and protease inhibitor cocktail (Sigma, Saint Louis, MO, USA. 1:1000). PP2Ac in cell lysates was immunoprecipitated with antibodies to PP2Ac (Millipore, Temecula, CA, USA), and protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Next, the beads were washed three times with the above lysis buffer, followed by washing twice with the phosphatase assay buffer (50 mM Tris-HCl, pH 7.0, 0.1 mM CaCl2). The phosphatase activity of immunoprecipitated PP2A was assayed with a Ser/Thr Phosphatase Assay kit 1 using p-nitrophenyl phosphate (pNPP) as the substrate (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. Finally, all data pooled from three different batches of experiments were statistically analyzed.