Fig. 3. Phosphorylation and accumulation of Smad2 occur rapidly and in proportion to activin concentration. (A) Phosphorylation of Smad2 can be detected 20 minutes after activin treatment. Dissociated animal cap cells from GST-Smad2-injected embryos were treated with activin (3 ng/ml) for 15 minutes, reaggregated, and cultured for the indicated length of time. GST pulled down proteins from the cell lysates were resolved on SDS-PAGE and transferred to nitrocellulose. The membrane was first blotted with an anti-phosphoserine antibody (top panel) to detect the phosphorylation of Smad2 and then blotted with an anti-GST antibody (bottom panel) to visualise total GST-Smad2. (B) GFP-Smad2 quickly enters the nucleus in cells treated with activin. Dissociated animal cap cells from GFP-Smad2-injected embryos were loaded onto a fibronectin substrate. When control embryos reached the indicated stage, the activin (5 ng/ml) was added to cells directly on the slides, and movement of GFP-Smad2 was observed by confocal microscopy in real time. (C) The phosphorylation of Smad2 is activin dose dependent. Dissociated animal cap cells from Myc-Smad2-injected embryos were treated with increasing doses of activin for 15 minutes and cultured in medium supplemented with 32P-orthophosphate, until stage 10.5. Cells lysates were subjected to immunoprecipitation with anti-Myc antibody. Immunoprecipitated proteins were resolved on SDS-PAGE and transferred to nitrocellulose. The phosphorylation of Smad2 was detected by autoradiography (top panel). The membrane was then blotted with an anti-Myc antibody (bottom panel) to visualise total Myc-Smad2. The Smad2 phosphorylation was quantitated and the values plotted. (D) The nuclear accumulation of Smad2 is activin concentration related. Dissociated animal cap cells from GFP-Smad2-injected embryos were treated with increasing concentrations of activin at the indicated stages, and loaded onto a fibronectin substrate. Thirty minutes later, the localisation of GFP-Smad2 was analysed by confocal microscopy. The nuclear GFP-Smad2 concentration was quantitated using the public-domain NIH Image program (Teleman and Cohen, 2000) and the values are shown at the bottom.
