Xenbase Image ID: 77657

Fig. 5. TOPFLASH reporter gene assay in Xenopus gastrula embryos. The TOPFLASH reporter vector (TOP, 200 pg), FOPFLASH vector (FOP, 200 pg), Flag-tagged β-catenin RNA (β-cateninFT, 300 ng), β-catenin RNA (β-catenin, 300 ng), Myc-tagged Zic RNA (Zic1–Zic5, 200 pg each), and/or Zic3 were injected into animal pole regions of both blastomeres of 2-cell-stage embryos in the indicated combinations. The TOPFLASH reporter vector contains Tcf-binding sites upstream of the promoter region, whereas the Tcf-binding sites are mutated in the control FOPFLASH vector. Embryos were collected at stage 10.5 in pools of 5 embryos, and assayed for luciferase activity in triplicate (total of 15 injected embryos for each value). The activity was normalized by the protein amount in the sample lysate. Mean luciferase activities of embryos injected with the TOP or FOP reporter vector only are defined as one relative luciferase unit (RLU) in each experiment. Error bars indicate standard deviation. (A) Co-injected β-catenin increased luciferase activity in the presence of the TOP reporter (TOP + β-cat). The increase was attenuated by Zic3 RNA co-injection (TOP + β-cat + Zic3). (B) This inhibition was rescued by co-injection of Zic3MO (Z3MO). (C) Comparison among the Xenopus Zic family members for the β-catenin-suppressing activities. (D) The effects of nuclear localization extent and transcriptional activation abilities of Zic3 or Zic2 mutants. Approximate nuclear localization extents and transcriptional activation abilities are indicated as follows: +++, ≥ 100; ++, 80–60; +, 50–30; ±, ≤ 20; where values of wild type Zic2 or Zic3 are 100. The values are based on previous studies [ZIC3-P217A, ZIC3-T323M, and ZIC3-K405E (Ware et al., 2004); ZIC3-W255G (Hatayama et al., 2008); Zic2-R428P (Hatayama et al., 2011)]. Image published in: Fujimi TJ et al. (2012) Copyright © 2012. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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