Fluorescence polarization of synthetic TRIB1 and C/EBPα peptides bearing an N-terminal FITC fluorophore (Mimotopes) was measured as indicated in combination with purified COP1 WD40 domain (376 to 731), TRIB1 (84 to 372), TRIB2 (53 to 343), or C/EBPβ degron peptide. COP1-TRIB1 and TRIB1/2-C/EBPα KD (dissociation constant) determination was performed in black 384-well microplates (Greiner Bio-One) with a final reaction volume of 30 μl, whereas TRIB1-C/EBPα displacement by C/EBPβ was performed in 96-well format with a final reaction volume of 60 μl. For COP1 displacement measurements, the COP1 WD40 domain and TRIB1-FITC peptide (349 to 367) were kept at a constant concentration of 1.5 μM and 25 nM, respectively, diluted in a fluorescence polarization buffer [300 mM NaCl, 10 mM Hepes, 0.5 mM TCEP, and 0.02% (v/v) Tween 20]. The concentrations of competing ligand, either TRIB1 protein or TRIB1 peptide, were varied from 0 to 20 μM. Where necessary, the reactions were supplemented with C/EBPα peptide (53 to 75) at a concentration of 10 μM. Once all reactions were prepared, they were allowed to incubate at room temperature for 20 min and measured using the POLARStar (BMG Tech) plate reader. Following this method, the data from three independent experiments were obtained and plotted as the means ± SEM using Prism 7. For TRIB1-C/EBPα displacement by C/EBPβ, 1.5 μM TRIB1 and 25 nM C/EBPα-FITC peptides were incubated together, C/EBPβ was titrated at concentrations from 43 nM to 350 μM, and measurement was performed in an equivalent manner.

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