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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.
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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.
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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).
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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”).
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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.
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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).
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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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