Recombinant IRF5 dimerization was measured by TR-FRET. Test peptides (2 mM stock in DMSO) were diluted threefold in DMSO and 2.5 μl per well added into 96-well polypropylene plates. Fifty microliters per well of 100 nM recombinant biotin-tag IRF5(222 to 467, S430D) and 250 nM recombinant His-tag IRF5(222 to 467, S430D) in Assay Buffer [50 mM tris-HCl (pH 7.4), 100 mM NaCl, 1 mM DTT, and BSA (0.2 mg/ml)] were added. Samples were incubated at RT for 20 min. Detection solution (17 μl per well) containing 10 nM europium-conjugated streptavidin and 80 nM allophycocyanin-conjugated anti–glutathione S-transferase antibody (Columbia Biosciences) in Assay Buffer (without DTT) were added. Samples were incubated at RT for 60 min followed by overnight incubation at 4°C, and 30 μl per well were transferred to 384-well polystyrene plates. Assay signals were monitored by reading excitation at 340 nm and emission fluorescence at 615 and 665 nm on an EnVision reader. Data were processed in Excel XLfit, and IC50 values were calculated using a nonlinear curve-fitting algorithm (four parameter equation; table S2).

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