Rra pt va

Threonine phosphatase-2A (PP2A) activity was measured using the Serine/Threonine Phosphatase Assay system (Promega; V2460) and the synthetic peptide RRA(pT)VA (Promega). For this assay, cells were lysed with Cellytic (Sigma) containing a protease inhibitor cocktail (Sigma), and the free phosphate was eliminated from the lysates using a Sephadex G-25 resin (Promega). For measurements of phosphatase activity, a standard phosphate curve was first constructed with 0, 100, 200, 500, 1,000 and 2,000 ρmol of phosphate. The samples (2.5 μg of total protein) were incubated with or without 12 nM or 5 μM okadaic acid for 15 minutes at room temperature. The reaction was performed by adding the PP2ase-2A 5× reaction buffer (250 mM imidazole, pH 7.2, 1 mM EGTA, 0.1% β-mercaptoethanol, 0.5 mg/ml bovine serum albumin) and the Thr phosphopeptide to the samples in a 96-well plate for 10 minutes at 30°C. The reaction was stopped by incubation with the molybdate dye for 15 minutes, and the absorbance was determined at 595 nm using a microplate reader (BioRad).