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	Figure 1. Identification of RanBP3 as an interaction partner of β-catenin. (A) Pull-down experiment using immobilized GST (lane 2), GST-tagged β-catenin ARM repeats 1–12 (lane 3), and full-length β-catenin (lane 4) incubated with X. laevis egg extract (input; lane 1). Bound proteins were analyzed by Western blot using mAb414 recognizing a subset of nucleoporins. Two unknown proteins, p80 and p90, are marked with arrows. (B) Identification of p80 and p90 as the b and a isoforms of RanBP3. Pull-down experiment as in A, incubated with HeLa nuclear extracts and analyzed using RanBP3 antibody. (C) RanBP3 binds directly to β-catenin. GST-tagged full-length β-catenin (lanes 2–11) was incubated with 2 μM RanGTP and 0.2 μM (lanes 2 and 5), 0.5 μM (lanes 3, 6, and 8–11), or 2.0 μM (lanes 4 and 7) wt (lanes 2–4 and 8–11) or wv mutant (lanes 5–7) RanBP3-b. Bound proteins were eluted as indicated above the lanes and visualized with silver (lanes 1–7) or Coomassie (lanes 8–11) staining.
	Figure 2. Expression of RanBP3 inhibits β-catenin/TCF–mediated transcriptional activation. (A) Wt and wv mutant RanBP3 are expressed at equal levels. HEK293 cells were transfected with the indicated constructs (ng), and lysates were analyzed 48 h after transfection by Western blot with the indicated antibodies. (B) RanBP3 represses Wnt1-induced β-catenin/TCF–mediated transcriptional activation dose dependently. HEK293 cells were transfected with TOP (black bars) or the control FOP (gray bars), Wnt1, and decreasing amounts of RanBP3 wt or wv mutant as indicated (ng), and luciferase activity was measured after 48 h. (C and D) RanBP3 represses transcriptional activation induced by wt β-catenin (C) or ΔGSK3–β-catenin (D). HEK293 cells were transfected with the indicated constructs, and luciferase activity was measured 48 h after transfection. In all experiments, normalized relative luciferase values are shown as corrected with pRL-CMV Renilla. Bars represent SEMs of independent experiments. (E) RanBP3 inhibits the expression of the endogenous Wnt target c-Myc. HCT116 colon carcinoma cells expressing Δ45–β-catenin were transfected with GFP and β-galactosidase, RanBP3 wt, or mutant plasmids. 2 d after transfection, GFP-positive cells were sorted using flow cytometry, lysed in sample buffer, and analyzed by Western blot using the indicated antibodies.
	Figure 3. Reduction of RanBP3 by RNAi results in increased β-catenin/TCF–mediated transcription activation. (A) Western blot showing that different shRNAs against RanBP3 reduce RanBP3 protein levels in HEK293 cells. Cells were transfected with shRNAs, and pHA262-PUR was cotransfected to introduce puromycin resistance. 24 h after transfection, cells were grown on puromycin medium for 48 h and cell lysates were prepared and analyzed on Western blot with the indicated antibodies. (B) RNAi against RanBP3 increases Wnt1-induced β-catenin/TCF–mediated transcription. HEK293 cells were transfected with the indicated constructs, and activity of TOP (black bars) and FOP (gray bars) was measured 72 h after transfection. Error bars represent SDs of technical replicates of a representative experiment. (C) RNAi against RanBP3 increases β-catenin/TCF–driven transcription in HEK293 cells that transiently express an active form of β-catenin (ΔGSK3–β-catenin). Cells were transfected with indicated constructs, and luciferase activity was measured after 72 h. (D) Coexpression of CRM1 with RanBP3 shRNA constructs does not affect β-catenin/TCF–mediated transcription in Wnt1-transfected cells. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection luciferase activity was measured. For all experiments, relative luciferase levels are shown as corrected with CMV-Renilla-luc. Error bars in C and D represent SEMs of independent experiments.
	Figure 4. RanBP3 antagonizes Wnt/β-catenin transactivation in APC-mutated colon carcinoma cells. Luciferase assay showing that RanBP3 inhibits β-catenin–mediated transactivation in colon carcinoma cell lines DLD1 and COLO320. (A) APC type I truncated human colon carcinoma cell line DLD1 (APC 1–1417) was transfected with luciferase reporter constructs and increasing amounts of RanBP3 expression constructs as indicated. DLD1 cells express a truncated APC protein that lacks all its COOH-terminal NESs. (B) Luciferase reporter assay as in A, performed in the APC type I truncated human colon carcinoma cell line COLO320 (APC 1–811). These cells express a short APC protein that lacks all β-catenin binding and regulatory sites. Relative luciferase activity was measured 48 h after transfection. Error bars show SDs of a representative experiment.
	Figure 5. Depletion of RanBP3 results in nuclear accumulation of active β-catenin. (A) Depletion of RanBP3 does not alter the levels of both total and active dephosphorylated β-catenin. HEK293 cells were transfected with or without Wnt1 and shRNA constructs against GFP or RanBP3. 72 h after transfection, whole cell lysates were analyzed by Western blot with the indicated antibodies. (B) RNAi against RanBP3 results in increased levels of active β-catenin in the nucleus. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection, nuclear and cytoplasmic extracts were prepared and analyzed by Western blot. TCF4 and tubulin staining are shown as markers for purity of the nuclear and cytoplasmic fractions. As a loading control in the nuclear fractions, TCF4 and a nonspecific reaction of the antibody recognizing active β-catenin are shown.
	Figure 6. RanBP3 induces specific depletion of endogenous nuclear active β-catenin. SW480 (A and B) or DLD1 (C) colon carcinoma cells were transfected with RanBP3 expression plasmids and stained after 45 h for dephosphorylated β-catenin (A and C) or total β-catenin (B). RanBP3 expression was visualized in the same cells using a RanBP3 polyclonal (A and C) or mAb. (D) Luciferase reporter assay as in Figs. 2–4 measuring relative β-catenin activity. Cells were transfected as in A and C. Error bars represent SDs of technical replicates.
	Figure 7. RanBP3 enhances nuclear export of active β-catenin independently of CRM1. (A and C) Effect of RanBP3 on mRFP–ΔGSK–β-catenin nucleocytoplasmic distribution in HEK293 cells in the presence or absence of 50 nM LMB for 3 h. (A) Box plot showing the distribution of nuclear/cytoplasmic ratios of mRFP–ΔGSK–β-catenin of two independent experiments. P values are according to Mann-Whitney tests. Representative mRFP fluorescence images are shown in C. Highlighted nuclear borders are drawn on the basis of accompanying phase-contrast images. (B) Functionality of mRFP–ΔGSK3–β-catenin. NCI-H28 cells (lacking endogenous β-catenin) were transfected with indicated constructs, and 48 h after transfection, luciferase activity was measured. Relative luciferase levels as corrected for transfection efficiency (Renilla luciferase activity) are shown. Error bars represent SDs. (D) Representative fluorescence images of HEK293 cells expressing GFP-Rev(1.4)-NES in the presence or absence of 50 nM LMB for 3 h. (E and F) Endogenous activated β-catenin relocalizes from the nucleus to the cytoplasm upon overexpression of RanBP3. HEK293 cells were transfected with Wnt and RanBP3 as indicated together with TOP-TK-luc and Renilla transcription reporter plasmids and fractionated after 48 h as in Fig. 5. Localization of active β-catenin was monitored using anti–active β-catenin antibody. Amounts of protein loaded were normalized on transfection efficiency (Renilla luciferase activity). Normalized β-catenin/TCF–dependent luciferase activity is depicted in F.
	Figure 8. RanBP3 rescues β-catenin–induced double axis formation in X. laevis embryos. (A) X. laevis embryos were injected ventrally at the four-cell stage with β-catenin mRNA in the presence or absence of control β-galactosidase or X. laevis RanBP3-b mRNA. (top) Wt noninjected embryos. (middle) Double axis phenotype as induced by the injection of β-catenin mRNA. (bottom) Embryos that are rescued from the double axis phenotype by coexpression of RanBP3 and β-catenin mRNA. (B) Quantification of the different phenotypes of two independent experiments in four categories: complete secondary axis (with cement gland), partial secondary axis (i.e., any secondary axis lacking the cement gland), vestigial axis (very small posterior protrusion or pigmented line), and normal (only one axis). P values are according to Pearson's χ2 test for count data. (C) Dorsal injection of RanBP3 results in ventralization of X. laevis embryos. Four-cell stage embryos were injected dorsally with RanBP3 or control (β-galactosidase) mRNA and analyzed 3 d later for ventralization using the standardized DAI. This scale runs from 0 (complete ventralization) to 5 (normal development). Frequencies are derived from three independent experiments. P values as in B. (D) The β-catenin downstream target siamois is significantly down-regulated in RanBP3-injected embryos. Embryos were injected as in C and analyzed for siamois or ornithine decarboxylase (ODC) mRNA using RT-PCR. Amplified ethidium bromide–stained DNA of four experiments was quantified and normalized to mean signals from β-galactosidase–injected embryos and represented in a box plot. P values are according to Mann-Whitney tests. (E) Representative signals from RT-PCR reactions visualized by ethidium bromide staining.
	Figure 9. Loss of RanBP3 by RNAi results in a naked cuticle phenotype in D. melanogaster. Shown are dark field images of cuticle preparations of control (β-galactosidase; A), D. melanogaster Daxin (B and C), and D. melanogaster RanBP3 dsRNA–injected embryos (D–F). Loss of Daxin and RanBP3 results in increased Wnt signaling and replacement of denticles by naked cuticle. Partially naked cuticles (B and D), nearly naked cuticles (E), and naked cuticles (C and F) are shown. All views are ventral, top is posterior. (G) Quantification of two representative experiments showing the frequency of the cuticle phenotype. P values are calculated as in Fig. 7 B. Note that the contribution of the completely naked phenotype in the RanBP3 RNAi embryos is relatively high (not depicted). (H) RT-PCR showing reduction in RanBP3 mRNA levels in RanBP3 dsRNA–injected embryos. Embryos were injected as in A, and RNA was extracted after 15 h of development. RT-PCRs specific for RanBP3 or control (ribosomal protein RP49) were performed using nothing (0) or a series of twofold dilutions of extracted RNA. (I) Loss of RanBP3 function by dsRNA injection results in increased expression of the wg target gene engrailed. Shown are an Engrailed antibody staining of a buffer-injected embryo (left), a Daxin dsRNA–injected embryo (middle), and an RanBP3 dsRNA–injected embryo (right). Note that the buffer-injected embryo developed until late stage 11, whereas the Daxin and RanBP3 RNAi embryos shown are stage 10 embryos, which explains the larger cells in the former embryo. The number of Engrailed-positive cell rows between stages 10 and 11 is identical. Ventral-lateral view is shown, posterior is left.

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