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Fig. 1. Mammalian Sprouty isoforms form homo-/hetero-oligomers through their C-terminal domains. (A) Expression constructs of Sprouty1- 4, either Flag-tagged at their N-terminus (F1-F4) or Myc-tagged at their C-terminus (1M-4M), are illustrated. Hatched boxes indicate cysteine-rich C-terminal domains. (B) 293T cells were co-transfected with two expression plasmids (0.5 µg each), one encoding one of the Flag-tagged Sprouty isoforms and the other encoding one of the Myc-tagged Sprouty isoforms as indicated. Cell lysates (500 µg protein) were subjected to immunoprecipitation (IP) using anti-Myc/anti-Flag antibody, followed by immunoblotting (IB) with anti-Flag/anti-Myc antibody. Rabbit non-immune IgG was used as a control. (C) F1, F1n, and F1c represent constructs encoding Flag-tagged full-length, N-terminal domain (residues 1-174), and C-terminal domain (residues 175-313) of Sprouty1, respectively. Expression of these constructs in 293T cells was assured by immunoblot analysis with anti-Flag antibody. (D) 293T cells were co-transfected with two plasmids (0.5 µg each), one encoding one of the Myc-tagged Sprouty isoforms and the other encoding Flag-tagged full-length, N-terminal domain or C-terminal domain of Sprouty1 as indicated. Cell lysates (500 µg protein) were subjected to immunoprecipitation using anti-Flag/anti-Myc antibody, followed by immunoblotting with anti-Myc/anti-Flag antibody. Data shown in B and D are representative of three separate experiments that gave essentially the same results.
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