4.5. Isothermal Titration Calorimetry

All ITC measurements were performed using NanoITC (TA Instruments, New Castle, DE, USA) at 15 °C. The ERK3 kinase domain in 20 mM HEPES, pH 7.5, 200 mM NaCl, 0.5 mM TCEP, and 5% glycerol at 180 µM was titrated into the reaction cell containing the inhibitors at 0.05—20.00 µM. The integrated heat of titration was calculated and fitted into an independent binding model using NanoAnalyzer software. Thermodynamic parameters, the dissociation constant (Kd), and stoichiometry (n) were an average over duplicates.