One microliter of test peptide (0.62 mM IRF5-CPP1 or 0.31 mM IRF5-CPP2-CPP6) was added into polypropylene 384-well microplates (Thermal Scientific). A 25 μl of 2.48 μM His-IRF5 (222 to 425) in Assay Buffer [20 mM tris-HCl (pH 7.4), 100 mM NaCl, and 1 mM dithiothreitol (DTT)] was added. Plates were centrifuged for 1 min at 1200 rpm (Eppendorf Centrifuge 5810 R) and incubated on a plate shaker at RT for 10 min. Five microliters of 25× Sypro Orange Dye (Invitrogen) diluted from the 5000× stock in Assay Buffer was added. After the plates were incubated at RT for 2 min, 20 μl per well of above reaction was transferred into Hard-Shell 384-well polymerase chain reaction (PCR) plates (Bio-Rad), followed by overlaying with 10 μl of mineral oil (Sigma-Aldrich) to prevent evaporation. The assay signals were monitored by reading excitation at 465 nm and emission fluorescence at 590 nm on a FluoDiaT70 reader (Photon Technology International) every 1.5°C increments from 30° to 55.5°C with a total of 18 reads. The fluorescence intensity signals fitted to Boltzmann equation were plotted using GraphPad Prism software.

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