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	FIGURE 1. ZIC2 negatively regulates the transcriptional activity of the β-catenin·TCF4 complex in 293T cells. A, activity of the TOPflash or FOPflash reporter in 293T cells transfected with ZIC2 expression vector in different amounts. B, TOPflash reporter activity of 293T cells transfected with ZIC2-expressing plasmid (20 ng) and increasing amounts of ΔN90-β-catenin-expressing plasmid (5–50 ng). The Western blot shows the levels of overexpressed proteins under each condition. C, TOPflash reporter activity of 293T cells treated with 20 mm LiCl for 12 h after transfection with the indicated plasmids.
	FIGURE 2. ZIC2 binding to TCF4. Cell lysates of 293T cells transfected with ZIC2-FLAG- and β-catenin-expressing plasmids together with TCF4-expressing vector (A) or without TCF4 (B) were subjected to immunoprecipitation (IP) using anti-FLAG antibody. C, schematic representation of LEFΔN-β-cateninΔN53 fusion protein. The graph shows the TOPflash reporter activity of 293T cells after transfection with the expression plasmids as indicated. D, Western blot showing the levels of the indicated proteins after FLAG-tagged ZIC2 overexpression in 293T cells.
	FIGURE 3. Mapping the ZIC2·TCF4 interaction domains. A, schematic representation of the different TCF4 mutants. The HMG domain and C-terminal region (CR) are represented as dark gray boxes. The β-catenin-interacting domain (β-Cat) is represented as a light gray box. B, Western blot showing transient coexpression of FLAG-tagged TCF4, deletion mutants, and ZIC2 using the indicated antibodies. C, immunoprecipitated proteins using anti-ZIC2 antibody (or IgG as a mock) were subjected to Western blot analysis for FLAG detection. D, immunoblot of in vitro protein binding assay. FLAG-tagged TCF4 and its truncated forms were purified with anti-FLAG beads (with IgG serving as a mock) and incubated with in vitro translated ZIC2, and the precipitated complex was subsequently subjected to immunoblotting using antibody against ZIC2. E, schematic representation of ZIC2 and its deletion constructs. Zinc finger domains are represented as dark gray boxes. F indicates FLAG. F, Western blot showing the protein levels of overexpressed full-length ZIC2, ZIC2 deletions, and TCF4 using the indicated antibodies. G, proteins immunoprecipitated from the lysates in F using anti-FLAG antibody (or IgG as a mock) were analyzed by Western blotting using anti-TCF4 antibody.
	FIGURE 4. Complex of ZIC2·TCF4 bound on DNA. A, EMSA was done by incubating a cell extract of 293T cells transfected with different expression plasmids as indicated with biotin-labeled probes containing wild-type or mutant TCF4-binding sequence. The lower panels represent Western blots of the corresponding samples as indicated. B, EMSA using 293T cells cotransfected with full-length TCF4 and its deletions together with ZIC2-expressing plasmid. Where indicated, the samples were preincubated with antibody against TCF4. C, DNA affinity precipitation of 293T cells transfected with the indicated expression vectors was performed as described under “Experimental Procedures.” The lysates were incubated with wild-type or mutant biotinylated probes corresponding to the TCF4-binding sequence, and the precipitated DNA·protein complexes were analyzed by Western blotting using the indicated antibodies. A probe with a mutant TCF-binding sequence was used as a control. D, 293T cells were transfected with ZIC2 and TCF4, and ChIP was performed with the indicated antibodies, followed by quantitative PCR using primer pairs spanning the human AXIN2 TCF4-binding site or an unrelated AXIN2 sequence as negative control. The ApoE promoter served as a positive control for ZIC2 binding. Results are presented as percentage of immunoprecipitated DNA over the input. E, luciferase reporter activity of the Wnt reporter (TOPflash) in 293T cells transfected with the expression vectors as indicated along with the β-catenin (DN90) expression vector.
	FIGURE 6. Zic2 morphants display increased Wnt reporter activity in the brain. Lateral views of stage 43 tadpoles injected with 40 ng of mismatch morpholino (MO) show normal development (A) and modest GFP expression (B). Embryos injected with Zic2 morpholino displayed loss of pigmented cells in the head and trunk (C) and markedly increased GFP expression (D), especially in the forebrain (open white arrowhead), the midbrain-hindbrain boundary (arrow), the dorsal side of the hindbrain (closed white arrowhead), and the eyes (closed yellow arrowhead). Note that the intestine shows autofluorescence (asterisk). The left-most tadpole is non-transgenic (NT). E–G, embryo injected unilaterally with Zic2 morpholino together with a red fluorescent LYSAMINE-labeled control morpholino. The injected side shows a marked increase in TOP-GFP expression, especially visible in the midbrain-hindbrain boundary (arrow).

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