In vitro binding assay was performed, as previously described (42). β-Catenin and Tcf7l2 were cloned into pET28a(+) vector and expressed in BL21(DE3) cells. His-tagged recombinant proteins were purified with Ni-NTA agarose resin (Qiagen) following the manufacturer’s instruction. In vitro translation of Flag-Zic2 or Flag-Zic5 was carried out using T7 Quick Coupled Translation/Transcription system (Promega) following the manufacture’s instruction. The FLAG-tagged proteins were immunoprecipitated with anti-FLAG M2 agarose beads (Sigma-Aldrich), followed by incubation with bacterially expressed His-tag proteins in binding buffer (1× phosphate-buffered saline, 0.1% NP40, 0.5 mM DTT, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride) for 4 hours at 4°C. The beads were washed for four times using ice-cold wash buffer and eluted with excess 3×Flag peptides (APExBio). Samples were boiled with SDS loading buffer before loading for SDS-PAGE.

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