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Fig. 2. Tagged forms of Smad2 and Smad4 retain their biological activities.
(A) Illustration of the principle of bimolecular fluorescence. In
unstimulated cells, in which Smad2 is not phosphorylated, Smad2 and Smad4 do
not interact, and the N-terminal and C-terminal portions of VENUS are not able
to complement. Phosphorylation of Smad2 results in a conformational change and
allows interaction with Smad4 and fluorescence complementation. (B)
Tagged Smad constructs retain their biological activity. Xenopus
embryos received no injections (embs, caps), or were injected at the one cell
stage with RNA encoding tagged forms of Smad2 or Smad4 proteins (100 pg for
VC155-Smad2/VNm9-Smad4 BiFC tags and 80 pg for HA tags), alone or in
combination, or with 5 pg RNA encoding Activin. Animal caps were dissected at
the midblastula stage and cultured to the equivalent of early gastrula stage
10.5. Note that when expressed individually, tagged forms of Smads do not
induce expression of Xbra and gsc, but co-expression of
Smad2 and Smad4 (100 pg RNA of each) activates both genes at levels similar to
those induced by Activin.
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