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	Figure 1. Immunochemical property of antigen of anti-Epith-1 mAb and -2 mAb in T. hardwicki using isoelectric points and molecular mass in Daltons separation (ISO-DALT) 2D immunoblotting and immunocrossreactivity of these two mAbs among seven species of sea urchins. (A) ISO-DALT 2D immunoblotting with anti-Epith-1 mAb shows an immunopositive spot at 160 kDa region and pH 4.98 (blue). The spot was artificially colored. (B) ISO-DALT 2D immunoblotting with anti-Epith-2 mAb shows an immunopositive spot (red) at the same region as (A). The spot was artificially colored. (C) Merged image between (A,B). (D) Immunocrossreactivity of anti-Epith-1 mAb (lanes 1–7) and anti-Epith-2 mAb (lanes 8–14). Lanes 1, 8; T. hardwicki (Th) swimming blastulae, lanes 2, 9; H. pulcherrimus (Hp) swimming blastulae, lanes 3, 10; C. japonicus (Cj) swimming blastulae, lanes 4, 11; M. globules (Mg) swimming blastulae, lanes 5, 12; L. pictus (Lp) swimming blastulae, lanes 6, 13; P. depressus (Pd) gastrulae, lanes 7, 14; S. intermedius mesenchyme blastulae (Si). Arrows, 160 kDa region. Arrowhead-1, 143 kDa region. Arrowhead-2, 137 kDa region.
	Figure 2. Isoelectric points and molecular mass in Daltons separation (ISO-DALT) analysis of Epith-2 of H. pulcherrimus (A–C) and proteinase sensitivity of Epith-2 T. hardwicki (D). (A) ISO-DALT 2D immunoblotting shows an immunopositive spot at the 143 kDa and pH 4.7 (red) region. The spot is artificially colored. (B) ISO-DALT 2D pattern stained with silver. (C) Merged image between (A,B). (D) Protease digestion of Epith-2 of swimming blastulae. Cont, control; the numbers, dilution ratios of protease, Tryp, trypsin digested. Chytrp, chymotrypsin digested.
	Figure 3. Immunochemical property of Epith-2 of H. pulcherrimus (A) and T. hardwicki (B) analyzed by immunoblotting. (A) Immunoblotting of Epith-2 extracted in water (lane 1) and in non-ionic detergent 1% Triton X-100 (lane 2) of swimming blastulae. Whole embryo lysate stained with concanavalin A (lane 3). (B) Mr of Epith-2 glycoprotein under non-reducing conditions (lane 1; 160 kDa region) and reducing conditions (lane 2; 174 kDa region).
	Figure 4. Immunochemical expression pattern of Epith-2 during early development of H. pulcherrimus. (A) Immunoblotting pattern. Lane 1; unfertilized eggs (uf), lane 2; fertilized eggs (f), lane 3; the 2-cell stage embryos (2cs), lane 4; the 4-cell stage embryos (4cs), lane 5; the 16-cell stage embryos (16cs), lane 6; morulae (Mor), lane 7; swimming blastulae (sBl), lane 8; mesenchyme blastulae (mBl), lane 9; mid-gastrulae (mG), lane 10; late gastrulae (lG), lane 11; prism larvae (Prsm), lane 12; pluteus larvae (Plut). Epith-2 is detected consistently at the 143 kDa region from unfertilized eggs to prism larvae, but a new band is detected at the 126 kDa region at the pluteus stage, which replaced the larger band of relative molecular mass. (B) Whole-mount double-stained immunohistochemical expression pattern of Epith-2 (green) and DNA with propidium iodide (red) (a–j’) and Polywax sagittal 6-μm thick sections (g”). (a) A 4-μm thick optical section of an unfertilized egg (uf). (a’) Higher magnification image of the box in (a). Arrow, anti-Epith-2 mAb-positive egg surface. (b) A 4-μm thick optical section of a fertilized egg (f). (b’) Higher magnification image of the box in (b). (c) A 4-μm thick optical section of a two-cell embryo (2cs). (c’) Higher magnification image of the box in (c). (d) A 4-μm thick optical section of a 4-cell embryo (4cs). (d’) Higher magnification image of the box in (d). Arrow, the basal surface of a blastomere. (e) Stacked image of a whole 16-cell stage embryo (16cs). (e’) A 3-μm thick optical section at the vegetal hemisphere. Arrow, the basal surface of a blastomere at the vegetal hemisphere. (e”) A 3-μm thick optical section at the animal hemisphere. Arrow, the basal surface of a blastomere. (f) Stacked image of a whole 32-cell embryos (32cs). (f’) A 3-μm thick optical section at the equator region. Arrow, the blastomere basal surface. (g) A 2-μm optical section of swimming blastula (sBl). (g’) Higher magnification image of the box in (g). (g”) A 6-μm thick sagittal Polywax section. (h) A 2-μm thick optical section of a mesenchyme blastula (mBl). (h’) Higher magnification image of the box in (h). Arrow, primary mesenchyme cells. (i) A 3-μm thick optical section of early mesenchyme blastula (eG). (i’) Higher magnification image of the box in (i). Arrow, primary mesenchyme cells. (j) A 3-μm thick optical section of late gastrula (lG). (j’) Higher magnification image of the box in (j). Arrows, secondary mesenchyme cells. Scale bars, 20 μm (a–j’), 50 μm (g”).
	Figure 5. Immunohistochemistry of Epith-2 internalization during mesenchyme ingression in H. pulcherrimus [(A–D); 6-μm thick Polywax sections], purified primary mesenchyme cells [PMC; (E,E’)] and the fate of Epith-2 in ingressed PMCs by immunoblotting in T. hardwicki (F,G). (A) An early mesenchyme blastula. Scale bar, 40 μm. (A’) Phase-contrast micrograph of the same section as (A). (B) Higher magnification image of the box in (A). Arrows, cytoplasmic anti-Epith-2 mAb-positive dots in primary mesenchyme cells (PMC). Scale bar, 10 μm. (C) Late gastrula. Scale bar, 40 μm. (C’) Phase-contrast micrograph of the same section as (C). Arrow, PMC aggregate near the blastopore. (D) Higher magnification image of the box in (C). Arrows, anti-Epith-2 mAb-positive dots in secondary mesenchyme cells (SMC). Scale bar, 10 μm. (E) Isolated PMCs stained with anti-P4 mAb. Scale bar, 30 μm. (E’) Phase-contrast micrograph of the same cells as (E). (F) A chart showing the sample preparation of the immunoblotting shown in (G). Samples in the broken-line box contain anti-P4 mAb and anti-mouse IgG Ab-tagged-magnetized Microbeads. Samples in the shaded box were examined with anti-Epith-2 mAb and anti-mouse IgG Ab. Samples in dark box were examined only with anti-mouse IgG Ab. (G) Epith-2 in the cytoplasm of PMCs analyzed with anti-Epith-2 mAb (lanes 1–6) and secondary antibody (Ab) alone (lanes 7, 8). Lane 1; anti-P4 mAb and anti-mouse IgG Ab-tagged-magnetized Microbeads-treated PMC fraction (PMCs), lane 2; anti-P4 mAb-treated epithelial cell fraction (Epithelial cells), lane 3; anti-P4 mAb-treated dissociated mesenchyme blastulae (Diss. Bl.), lane 4; whole mesenchyme blastulae (Whole mBl), lanes 5, 7; anti-P4 mAb alone (P4 mAb), lanes 6, 8; anti-mouse IgG Ab-tagged magnetic Microbeads alone (Microbeads). Arrows, Epith-2 at 160 kDa region. Arrowheads, IgG of primary Ab.
	Figure 6. Inhibition of Epith-2 endocytosis and PMC spreading by herbimycin A (HA) and suramin and the analysis of phosphorylation site of Epith-2 of H. pulcherrimus. (A) Immunohistochemistry of HA-treated mesenchyme blastula using 6-μm thick Polywax section. Red arrow, lack of Epith-2 on the cell surfaces of PMCs and neighboring epithelial cells. Scale bar, 40 μm. (B) Higher magnification of box in (A). Arrowheads, cell-surface-associated Epith-2. Arrows, dots of cytoplasmic Epith-2. Red arrow, lack of Epith-2 on the cell surface of PMC and neighboring epithelial cell. Scale bar, 20 μm. (C) Immunohistochemistry of control mesenchyme blastula using 6-μm thick Polywax section. Scale bar, 40 μm. (D) Higher magnification of the box in (C). Arrows, cytoplasmic Epith-2. Scale bar, 20 μm. (E) A 2-μm thick optical section of a confocal laser scanning micrograph of a suramin-treated early gastrula. Epith-2 expressed in an aggregate of PMCs near the archenteron [box (e’)]. Epith-2 (green) expressed in aggregated-PMCs on top of the archenteron [box (f)]. Scale bar, 40 μm. (E’) Higher magnification of the box (e’) in (E). Arrow, Epith-2 cell. Scale bar, 5 μm (F) Higher magnification image of the box (f) in (E). Arrow, aggregated PMCs. Scale bar, 20 μm. (G) Epith-2 phosphorylation in swimming blastulae by immunoblotting. Lane 1; anti-Epith-2 mAb, lane 2; anti-phosphotyrosine (PY) antibody (Ab), lane 3; anti-phosphoserine (PS) Ab, and lane 4; anti-phosphothreonine (PT) Ab. Arrow and dotted line denote the 143 kDa region. Inset; immunoblotting with antibodies against Epith-2, phosphotyrosine (PY), phosphoserine (PS), and phosphothreonine (PT) using single-lane SDS-PAGE gel. Arrow, 143 kDa region. (H) Phosphotyrosine detection by immunoblotting in Epith-2 of swimming blastulae by isoelectric points and molecular mass in Daltons. (a) Anti-Epith-2 mAb. Artificially colored with red. (b) Anti-PY Ab. Artificially colored with blue. Inset, higher magnification of tail region. Arrows, anti-PY Ab-positive dots. (c) Merged image between (a,b). Numbers on the top show the pH gradient from the left (4.7) to right (6.4).
	Figure 7. Embryonic cell reaggregation assay in the presence of IgG of anti-Epith-2 monoclonal antibody in H. pulcherrimus. (A) Whole embryonic cells of swimming blastulae immediately after dissociation (0 h). (B–D) Re-aggregated embryonic cells at 5 h after dissociation. (B) In plain artificial seawater (ASW alone). (C) With 10 μg/ml IgG (10 μg/ml IgG). (D) With 50 μg/ml IgG (50 μg/ml IgG). Scale bars, 100 μm. (E) Average size of cell aggregates with no IgG (0), 10 μg/ml (10) and 50 μg/ml (50) IgG. Bars, SD (n = 80). *P = 0.0344, **P = 0.0001. Unpaired t test. (F) Proportion of three cell aggregate sizes in 0 μg/ml (0), 10 μg/ml (10), and 50 μg/ml (50) anti-Epith-2 mAb IgG. Bars, SD (n = 80). *P = 0.0055, **P = 0.0588. Unpaired t test.

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