Additional Supporting Information may be found in the online version of this article.

DVDY_22125_sm_suppinfofigure1.tif2209KFig. S1. Characterization of small deletions in the XARP C-terminus. A–E: Unlike deletion K4 (Fig. 1F), small deletions K1, K2, and K5 in the context of full-length GFP-XARP do not abrogate its centrosomal localization. Deletions K3 and K6 just upstream of the DIX domain appear to localize to the cytoplasm. Staining was done as in Figure 1. F,G: By Western blot analysis, all small deletions, except for K3 and K6, are well expressed after injecting 2 ng mRNA. GFP-K3 and GFP-K6 cannot be detected at any dose (F), while myc-K3 and myc-K6 after injecting 15 ng mRNA are detected as a series of degradation products (G), which likely explains their abnormal localization (C,E). β-tubulin served as a loading control. The following amino acids were removed by deletions: K1, 540–559; K2, 562–573; K3, 631–648; K4, stop-codon after aa651; K5, 533–593; K6, 610–645.
DVDY_22125_sm_suppinfofigure2.tif4497KFig. S2. A: Subcellular localization of myc-wild-type XARP and m5-m8 detected by mouse anti-myc antibody, followed by goat Cy3-anti-mouse IgG antibody. B: Western blot analysis confirming stability of constructs m5-m8 (β-tubulin served as a loading control). C–G: Examples of sections that have indicated ratios of cells with centrosomal staining. In parentheses, injected constructs are indicated. Staining was done as in Figure 1; the presence of the centrosomes was confirmed by γ-tubulin co-staining (not shown).
DVDY_22125_sm_suppinfofigure3.tif6470KFig. S3. Embryo ventralization assay. Xenopus embryos were injected into the dorsal marginal zone at the 4-cell stage with 0.8 ng RNA encoding myc-wild-type XARP, m8, or K4, and, at stage 28, classified as either normal, with short axis, or without axis. Some embryos were used for accompanying Western blot analysis. Embryos representing each phenotype, along with injected constructs in parentheses, are shown on the right. Numbers above graph bars represent total number of analyzed embryos from two experiments, except for m8 (one experiment).
DVDY_22125_sm_suppinfofigure4.tif1232KFig. S4. XARP localization changes in response to Fz protein accumulation. A,B: Xenopus embryos were injected with GFP-XARP and Dsh RNAs and pCS2-myc-dFz1 DNA and stained at stages 9 and 11 with rabbit GFP antibody (green) and mouse anti-myc antibody (red). pCS2-myc-dFz1 plasmid encodes myc-dFz1 under the eukaryotic promoter CMV and is silent until midblastula transition (Heasman, 2006). Concomitantly with accumulation of Fz protein between stages 9 and 11, GFP-XARP appears at the plasma membrane. Since no detectable novel synthesis of GFP-XARP occurs between stages 9 and 11 (C, β-tubulin is the loading control), it is likely that previously made GFP-XARP protein dynamically changed its localization. Note that cells in B are smaller than in A because of several additional rounds of cell division.
DVDY_22125_sm_suppinfofigure5.tif2731KFig. S5. Subcellular localization of GFP-XARP in the absence or presence of HA-XARP-C (controls for Fig. 6F). After injection of indicated RNA combinations, embryos were cryosectioned at gastrula stage 10 and stained as described in Figure1, except that mouse-HA antibody was used in E. Bottom panels are merged images of the top two panels.
DVDY_22125_sm_suppinfofigure6.tif4396KSupporting Information Figure 6

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