Kinetic Assay of IKKα Activity

IKK $\alpha$ kinase activity was investigated performing the reactions at 30 °C for 15′, 30′, 1 h, 2 h, 4 h, and 6 h in a volume of 10 µL in Eppendorf^® PCR tubes. IKK $\alpha$ recombinant enzyme (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) at the final concentration of 0.0607 µg/µL was diluted in a reaction buffer containing 50 mM Tris-HCl, 0.1 M NaCl, 5 mM MgCl₂ and 1 mM DTT (all purchased from Sigma Aldrich, Co. Saint Louis, MO, USA), in presence of 500 µm Mg²⁺-ATP (Sigma Aldrich) and 0.2 µg/µL IKKtide (Promega Corporation, Madison, WI, USA). The K_m value was determined by assaying enzyme activity using the peptide in the range 0.0175–0.175 mM. The amount of IKK $\alpha$ to be used was chosen on the basis of the specific activity declared by the manufacturer. The amount of IKKtide was suggested by the manufacturer’s instructions, finally, the best concentration of ATP was determined in the authors’ laboratory [20 (link)]. At the end of incubation time, each sample from all time points was diluted in a 10 µL solution of CH₃CN/H₂O (ratio 1:3) to stop the reaction, centrifuged at 14,000 rpm for 10’ to discard any impurities and then ultrapure water was added to the supernatant to reach a final volume of 30 µL, using 20 µL of these for the UPLC/MS analysis.