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Subsequently, a dual-luciferase reporter assay was performed by constructing pmirGLO dual-luciferase reporter vectors containing either the wild-type WNT3A 3UTR sequence (WT-WNT3A) or the mutated version of this sequence (MUT-WNT3A), to validate this interaction

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After the tags were removed, via proteolytic cleavage, the SKP1-FBXO22 and CUL1-RBX1 complexes were further purified using anion exchange chromatography and cation exchange chromatography, respectively, and then by size exclusion chromatography (SEC) into 20 mM HEPES pH 8, 200 mM NaCl, 1 mM DTT