In vitro phosphatase assay with pNPP

To test the phosphatase activity of the purified phosphatases, we performed an assay using the general substrate p-nitrophenyl phosphate (pNPP, New England Biolabs). The pNPP (50 mM) was incubated with INTAC complex, PP2A core enzyme, PP1(50 nM), or SSU72 (50 nM and 0.5 μM) in a final volume of 20 μl containing 50 mM HEPES pH 7.4, 100 mM NaCl, 0.01% CHAPS, 10 mM MgCl₂, 1 mM MnCl₂ (without MnCl₂ for SSU72 owing to the decreased activity in the presence of MnCl₂), and 2 mM DTT. After incubation at 30°C for 20 min, the reactions were stopped by adding 80 μl of 1 M NaOH, and the released pNPP was measured at optical density at 405 nm (OD 405 nm) using a Synergy H4 Hybrid Microplate Reader (BioTek Instruments). The experiments were performed in triplicates.