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Fig. 4. Treatment with ephrin-B1–Fc induces retraction of medullary tubule cells plated on Matrigel-coated surfaces. (A) Cells were stimulated with 1 µg/ml ephrin-B1–Fc or 1 µg/ml human Fc for the indicated time periods. Cell lysates were immunoprecipitated with anti-EphB2 antibody. The immunoprecipitates were separated by SDS-PAGE, probed by immunoblotting with phosphotyrosine (PY) antibodies, and reprobed for EphB2. (B,C) Phase-contrast time-lapse microscopy pictures at a low (B) and high (C) magnification. Cells were stimulated with 1 µg/ml ephrin-B1–Fc and a series of phase-contrast images of the same field were obtained at the indicated times. Treatment with ephrin-B1–Fc leads to dramatic changes in cell morphology. Cell retraction is particularly prominent at the free edges of the cells (arrowheads in B) and partial separation between the adjacent cell surfaces of neighboring cells is also evident (arrow in C). (D) Morphological changes are induced by ephrin-B1–Fc but not EphB2-Fc treatment. Cells were stimulated with ephrin-B1–Fc (1 µg/ml) or EphB2-Fc (1 µg/ml) with or without crosslinking with anti-human Fc antibody (0.25 µg/ml). 1 µg/ml Fc with or without anti-human Fc antibody, or vehicle only, were used as controls. Phase-contrast images of the same field were obtained at 0 minute and 15 minutes after beginning the stimulation and the fraction of retracting cells at the 15 minutes time point was calculated. Only cells at the periphery of the epithelial sheets were quantitated for statistical analysis. Data from three independent experiments are shown as mean ± s.d. Comparisons were performed using one-way factorial ANOVA.
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