Fig. 5. Nuclear Smad2 concentration is maintained by continuous degradation and nuclear entry. (A) Smad2 has a 2-3 hour half-life in activin-treated cells (graph). Dissociated animal cap cells were subjected to a pulse-chase metabolic labelling (with [³⁵S]methionine/cysteine), treated or not with activin, and Smad2 was immunoprecipitated with an anti-Smad2 antibody. The specificity of immunoprecipitation was confirmed by using protein-A-sepharose alone (data not shown). (B) Activated Smad2 has a 1-1.5 hour half-life (graph). Dissociated animal cap cells were subjected to a pulse-chase metabolic labelling (with [³²P]orthophosphate), treated with activin, and Smad2 was immunoprecipitated with an anti-Smad2 antibody. Un, untreated. (C) Phosphorylation of Smad2 is detected long after activin treatment. Dissociated animal cap cells were treated with activin for 15 minutes and cultured in medium supplemented with [³²P]orthophosphate either just after or 1.5 hours after activin treatment. Smad2 was immunoprecipitated with an anti-Smad2 antibody. (D) GR-GFP-Smad2 fusion construct. (E) Dexamethasone treatment can induce nuclear Smad2 localisation, several hours after activin has been removed. Dissociated animal cap cells from GR-GFP-Smad2-injected embryos were treated with activin for 15 minutes, extensively washed, loaded onto a fibronectin substrate before control embryos reached stage 9. Three hours after stage 9, GR-GFP-Smad2 release was induced by addition of dexamethasone and GR-GFP-Smad2 localisation observed by confocal microscopy in real time.