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Fig. S5 Zebrafish angiopoietin 2a binds to human TIE-2 receptor. (A) A comparison of zebrafish angiopoietin 1 and 2 and their orthologues in Fugu and Human. Clustal W alignment with the protein sequences of zebrafish (Danio rerio, Drer), Fugu-Fish (Takifugu rupripes, Trub) and of human (Homo sapiens, Hsap) shows that zebrafish and fugu-fish angiopoietin 1a and 1b are grouped together with human Angiopoietin 1. Zebrafish and fugu-fish angiopoietin 2b are clustered together with human Angiopoietin 2, whereas zebrafish and fugu-fish angiopoietin 2a is clustered as a separate group. (B) Zebrafish angiopoietin 2a (ang2a) binds to human Tie2-Fc. Full length zf ang2a cDNA was cloned in pCDNA3.1. ang2a, CA1 (Comp-Ang1) or empty pCDNA3.1 as a control were transiently expressed and 35S labelled in 293T cells. hTie2 protein (R&D) were used to pull-down 35S labeled angiopoietins from 293T conditioned medium. The bound proteins were separated on SDS-PAGE, and detected using autoradiography. The results show that hTie2-Fc binds to COMP-Ang1 as well as to Ang2a.