Figure 5.

(A) Pulldown experiment using cytoplasmic tails of zebrafish ephrinB2, ephrinB2 (4A), and integrin ?1A or ?IIb immobilized on NeutrAvidin beads with a cell lysate expressing GFP-zebrafish kindlin2. Bound proteins were fractionated on SDS?PAGE. His/Avi-tagged cytoplasmic tails were stained by Coomassie blue, and kindlin2 was recognized by Western blotting with anti-GFP antibody. Asterisk indicates the position of purified his/Avi-tagged cytoplasmic tails. (B) Percentage of zebrafish embryos injected with efnb2a WT or 4A RNAs displaying defects in blood flow. The data are presented as the mean ± SEM (n = 3). An unpaired two-tailed t test was used. **P < 0.01. (C, D, E) Some zebrafish embryos that overexpressed ephrinb2a displayed extremely narrow dorsal aorta (indicated by arrow) and inadequate intersegmental vessel growth (indicated by star), whereas embryos that overexpressed 4A mutant of ephrinb2a (D) and uninjected embryos (UIC) (E) did not show these defects (red bar indicated the 10 aorta, and arrowhead indicated the intersegmental vessel). (F, G, H) Other zebrafish embryos that overexpressed ephrinb2a failed the arterial/venous segregation (indicated by white bar) in local area of the trunk, whereas this defect was not seen in 4A-overexpressed embryos (G) and uninjected embryos (UIC) (H) (red and yellow bars indicated the dorsal aorta and posterior cardinal vein, respectively). Scale bar, 100 µm (C, D, E) and 50 ?m (F, G, H).

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