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. 2013 Mar 15;288(18):12469–12477. doi: 10.1074/jbc.M112.433979

FIGURE 4.

FIGURE 4.

YIA sequence of Tie2 induces ligand-independent dimerization. A, schematic of Tie2/Tie1* chimeras. B, in HEK293T cells, Tie2-HAVN was transiently transfected in combination with Tie2, Tie2(1088)Tie1*, Tie2(975)Tie1*, Tie2(932)Tie1*, Tie2(745)Tie1*, Tie2(212)Tie1*, or Tie1* C-terminally fused with MycVC, and close associations of receptors assessed by BiFC and flow cytometry (*, p < 0.05; n = 3). The protein expression level of each receptor was confirmed by immunoblotting with anti-HA or anti-Myc Ab (inset). C, comparison of amino acid sequences of Tie1 and Tie2 C terminus from different species. Pink, different amino acids; blue, same amino acids (aa). D and E, in HEK293T cells, Tie2-HAVN was transiently transfected in combination with Tie2, Tie2YIA/LAS, Tie2Q988E, Tie2N1017K, Tie2Y1023F, Tie2LEK/MEQ, Tie2L1060R, Tie2D1068E, Tie2EK/DR, Tie2S1083P, or Tie1* C-terminally fused with MycVC (D) or Tie2, Tie2Y975L, Tie2I976A, Tie2A977S, Tie2YIA/LAS, or Tie1* C-terminally fused with MycVC (E), and close associations of receptors assessed by BiFC and flow cytometry. Protein expression level of each receptor was confirmed by immunoblotting with anti-HA or anti-Myc Ab (inset) (*, p < 0.05; n = 3). F, YIA domain of Tie2 was replaced by LAS sequence (Tie2YIA/LAS). Association of Tie2-Tie2, Tie2-Tie2YIA/LAS, and Tie2YIA/LAS-Tie2YIA/LAS was observed by BiFC as described above. Protein expression level of each receptor was confirmed by immunoblotting with anti-HA or anti-Myc Ab (inset) (*, p < 0.05; n = 3).