Journal: Developmental biology

Article Title: Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin.

doi: 10.1016/j.ydbio.2004.03.043

Figure Lengend Snippet: Fig. 5. Ultrastructural immunolocalization of p160 (using the antipeptide antibody) shows its enrichment at the tips of the microvilli in unfertilized eggs (A, B), a transient presence in the forming fertilization envelope (C), and a lack of p160 signal in the mature fertilization envelope (D). CG: cortical granules; MV: microvilli. Scale bar = 0.25 Am.

Article Snippet: Primary antipeptide antiserum was diluted 1:25,000 and the secondary antibody (Cy3-conjugated affinity-purified goat antirabbit IgG; Jackson Immunoresearch, West Grove, PA) was diluted 1:200.

Techniques:

Journal: Developmental biology

Article Title: Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin.

doi: 10.1016/j.ydbio.2004.03.043

Figure Lengend Snippet: Fig. 3. p160 is proteolyzed and released from the cell surface at fertilization. Two different preparations of exudate released following fertilization were stained with Coomassie (lanes 1, 2). These samples were then immuno- blotted against antipeptide preimmune serum (lanes 3–5), antipeptide serum (antipeptide; lanes 6, 7), antipan p160 serum (lanes 8, 9, shown from two different gels). The antibodies detect an overlapping set of p160 fragments at 85, 60, and 35 kDa. Samples were also immunoprecipitated with either antipan p160 (lanes 10, 11) or preimmune serum (lanes 12, 13). The starting samples of two different biotinylated exudates used in the immunoprecip- itation experiments are shown (lanes 14, 15). Note that the p160 immuno- blot results recapitulate the immunoprecipitation results.

Article Snippet: Primary antipeptide antiserum was diluted 1:25,000 and the secondary antibody (Cy3-conjugated affinity-purified goat antirabbit IgG; Jackson Immunoresearch, West Grove, PA) was diluted 1:200.

Techniques: Staining, Immunoprecipitation

Journal: Developmental biology

Article Title: Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin.

doi: 10.1016/j.ydbio.2004.03.043

Figure Lengend Snippet: Fig. 4. p160 is on the egg cell surface, and is removed at fertilization. Sections of fixed unfertilized and fertilized eggs were immunolabeled with the antipan p160 antibody. (A, B) Eggs; (C, D) 5 min post fertilization (FE denotes fertilization envelope, which is misshapened during processing); (E, F) immunolabeling of eggs in which the antipeptide antibody was preincubated with the immune peptide. Scale bar = 40 Am.

Article Snippet: Primary antipeptide antiserum was diluted 1:25,000 and the secondary antibody (Cy3-conjugated affinity-purified goat antirabbit IgG; Jackson Immunoresearch, West Grove, PA) was diluted 1:200.

Techniques: Immunolabeling

Journal: Developmental biology

Article Title: Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin.

doi: 10.1016/j.ydbio.2004.03.043

Figure Lengend Snippet: Fig. 7. p160 is enriched in the egg plasma membrane. Immunoblot analyses of different egg fractions probed with antipeptide antibody. Lane 1: whole egg lysate; lane 2: cell surface complex; lanes 3 and 4: different preparations of egg plasma membrane. Five micrograms of each preparation was loaded in each lane. The arrow indicates the band of 160 kDa.

Article Snippet: Primary antipeptide antiserum was diluted 1:25,000 and the secondary antibody (Cy3-conjugated affinity-purified goat antirabbit IgG; Jackson Immunoresearch, West Grove, PA) was diluted 1:200.

Techniques: Clinical Proteomics, Membrane, Western Blot

Journal: Developmental biology

Article Title: Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin.

doi: 10.1016/j.ydbio.2004.03.043

Figure Lengend Snippet: Fig. 8. p160 removal from the egg surface is serine protease dependent. Epifluorescence images of live eggs immunolabeled with the antipeptide antibody: (A) p160-labeled control eggs and (B) after 10 min post activation with A23187. (C) p160-labeled eggs, incubated in SBTI before activation. (D) In the presence of SBTI, p160 label is not removed from activated eggs, up to 10 min post activation. Scale bar = 40 Am.

Article Snippet: Primary antipeptide antiserum was diluted 1:25,000 and the secondary antibody (Cy3-conjugated affinity-purified goat antirabbit IgG; Jackson Immunoresearch, West Grove, PA) was diluted 1:200.

Techniques: Immunolabeling, Labeling, Control, Activation Assay, Incubation

Journal: Developmental biology

Article Title: Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin.

doi: 10.1016/j.ydbio.2004.03.043

Figure Lengend Snippet: Fig. 11. Proper formation of the fertilization envelope is prevented by either protease inhibitors or by antip160 antibodies upon activation by A23187. (A, B) Antipan p160 whole antibody; (C, D) antipan p160 Fab; (E, F) antipeptide Fab; (G, H) SBTI. Scale bar = 20 Am.

Article Snippet: Primary antipeptide antiserum was diluted 1:25,000 and the secondary antibody (Cy3-conjugated affinity-purified goat antirabbit IgG; Jackson Immunoresearch, West Grove, PA) was diluted 1:200.

Techniques: Activation Assay