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Fig. 2 Zebrafish Tie1 (zTie1) acts as the receptor for zAngpt1. (A) Schematic of the in vitro binding assay for B. The detailed procedure is described in the Materials and Methods section. Light blue circle, protein G sepharose beads. (B) In vitro binding of zebrafish Tie1 (zTie1)-Fc-His, zTie2-Fc-His or Fc-His to FLAG-Angpt1, FLAG-Angpt2a or FLAG-Angpt2b was examined. (C) 293T cells transfected with full-length zTie1 (zTie1-HA) and a kinase-deficient mutant of zTie1 (zTie1K854R-HA) were starved and stimulated with vehicle (−) and COMP-Ang1 (+) for 20 min. Cell lysates were subjected to immunoblot analyses with anti-phosphotyrosine (pTyr) and anti-HA antibodies for analyzing phosphorylated Tie1 and total Tie1, respectively, and also with anti-β-actin antibody. (D) Relative phosphorylation of zTie1 observed in C was quantified. Data are mean±s.d. from three independent experiments; each experiment includes duplicate determinations. P-values were determined by two-tailed Student's t-test.