Lim JC , Kurihara S , Tamaki R , Mashima Y , Maéno M .

	Fig 1. Biochemical properties of Rdd proteins. (A) rdd3-ha mRNA was injected into 2-cell-stage embryos. The embryos were cultured until the late neurula stage. Western blot analysis was performed with extracted proteins after treatment with N-glycosidase F (lane 2) or no treatment (lane 1). The signal of Rdd3-HA was shifted by N-glycosidase F treatment, indicating that recombinant Rdd3 protein was N-glycosylated. (B) Western blot analysis was performed with extracted proteins from uninjected or rdd4-flag mRNA-injected embryos under the reducing (lanes 1 and 2) or non-reducing (lanes 3 and 4) condition. Rdd4-Flag appeared as a single band under the reducing condition (lane 2), but it showed a smear at the high molecular weight range under the non-reducing condition (lane 4).
	Fig 2. Localization of recombinant Rdd3 protein in the intercellular region. val-flag mRNA (Flag-tagged Val protein) (A-C) or rdd3-flag mRNA (Flag-tagged Rdd3 protein) (D-I) together with mif-myc mRNA (coding for Myc-tagged Mif protein) were injected into a single blastomere at the 16-cell stage. Animal cap explants were isolated and cultured until the early tailbud stage (st. 20). Explants were stained with anti-Myc and anti-Flag antibodies simultaneously and the reaction was visualized with Alexa488-based fluorescence (A, D, H) and Cy3-based fluorescence (B, E, G). Positive signal for Mif protein shows descendant cells of the RNA-injected single cell at the 16-cell stage (A, D, G). The border between the injected and uninjected cells is indicated by a white dotted line. Merged images of the double staining are also shown (C, F, I). Val-Flag protein was detected in the nuclei of cells that had been injected with mif-myc mRNA (C, C'). On the other hand, Rdd3-Flag protein was stained in the cytoplasm of Mif-positive cells and also stained in the intercellular region of Mif-negative cells (F, F'). Exchange of the second antibodies between Cy3-conjugated IgG and Alexa488-conjugated IgG gave the same results (I, I'). Scale bars in panels I and I'=50 µm (A-I), 50 µm (C', F', I').
	Fig. 3. Production of anti-sera against a synthetic peptide from the Rdd3 amino acid sequence. (A) Western blot analysis was performed with anti-HA antibody or anti-Rdd antibody to detect HA-tagged recombinant Rdd proteins. Anti-HA antibody reacted with Rdd2, Rdd3, and Rdd4 proteins (lanes 1-3), while anti-Rdd antibody reacted with Rdd3 and Rdd4 (lanes 5 and 6). (B) Absorption of anti-serum with Rdd (lane2) or control (lane 3) peptide indicated specific reaction of the antibody with Rdd4 protein in Western blot analysis.
	Fig 5. Concomitant localization of Rdd3 protein with blood vessel formation. Whole-mount in situ hybridization (A) and whole-mount immunostaining (B) analyses were performed to show tie-2 expression and Rdd protein localization in tailbud (st. 32) embryos. Embryos were injected with vascular endothelial growth factor Morpholino oligo (VEGF MO) (9.2 pmol/embryo) (d-f ) or H2O (a-c) in the marginal zone at the 4-cell stage. (A) In the control embryo, tie-2 was expressed in the vitelline vein (b, arrowheads), intermediate mesoderm (c, white arrowheads) and branchial arches (c, arrowheads). Localization of tie-2 expression was lost in the VEGF MO-injected embryo (d-f ). (B) Rdd protein was detected in the vitelline vein (b, arrowheads), intersomite region (c, white arrowheads), and branchial arches (c, arrowheads). Localization of Rdd protein was lost in the VEGF MO-injected embryo (d-f ). Scale bars=0.5 mm (A, B).

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