2.8. Calcineurin activity assay

Calcineurin (PPase‐2B, CaN) was purchased from Promega (V6361, isolated from bovine brain). Dephosphorylation of the substrate p‐nitrophenyl phosphate (PNPP, Sigma, 1040) was followed by monitoring the increase in absorbance at 405 nm using a Perkin Elmer EnSpire microplate reader. The CaN activity was assayed by measuring the initial velocities at 28°C in 50 mmol/L Tris, pH 7.5 containing 0.001 unit/μL CaN, 0.1 mg/mL BSA, 20 mmol/L PNPP, 1 mmol/L NiCl₂, and various amounts of CaM to determine dose‐response curves for CaN activation by CaM. For testing inhibition of CaM function, 10 nmol/L CaM and various amounts of the small molecules were used.