Measurement of calcineurin phosphatase activity

Calcineurin activity was determined using a colorimetric assay as per manufacturer’s instructions (Enzo Life Sciences). HEK293T cells were seeded onto 60 mm culture dishes at a density of 10⁶ per dish and tested 24 h later. Following treatment, cells were washed twice with ice-cold Tris buffered saline solution (20 mM Tris, 150 mM NaCl, pH 7.2) and lysed in a solution containing (in mM); 50 Tris, 0.1 EDTA, 0.1 EGTA, 1 DTT, 0.2% NP-40, pH 7.5 with a protease inhibitor tablet and stored at −80 °C. Excess phosphates and nucleotides were removed from the lysates by passing the samples through a chromatography column and the desalted samples were stored at −80 °C. To ensure an equal amount of protein was run in the assay for each sample, a bicinchoninic acid (BCA) assay was run according manufacturer’s instructions. 3 µg of protein per sample was used for the calcineurin phosphatase assay. Total phosphatase activity in the samples was detected by addition of the phosphopeptide substrate, RII, in assay buffer. The assay plate was then equilibrated to the reaction temperature of 37 °C for 10 min and sample lysates were added to the assay plate at 37 °C for 30 min. The free phosphate was then measured by the addition of Biomol Green reagent and colour was allowed to develop for 30 min at 37 °C. Absorbance was measured at 620 nm and data were background corrected.