Fig. 8. The receptor complex remains activated after a short exposure to activin. (A) Gene expression is blocked by serine/threonine kinase inhibitor H7. RNase protection analysis for Apod, Eomes and Xbra expression, in activin-induced cells treated with the indicated concentration of inhibitor H7. WE, whole embryos. (B) H7 inhibits phosphorylation on the C-terminal serine of Smad2. Dissociated animal cap cells from GST-Smad2-injected embryos were treated with activin for 15 minutes, reaggregated and cultured minus or plus H7 inhibitor, at the indicated concentration, until stage 10.5. 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. (C) H7 blocks gene activation by a constitutively active type I receptor (Alk4*), whereas gene activation by a constitutively phosphorylated Smad2 (Smad2*) is not affected by H7. RNase protection analysis for Apod, Eomes and Xbra expression, in animal cap cells from Smad2*- or Alk4*-injected embryos, treated with the indicated concentration of inhibitor H7. WE, whole embryos. (D) Delayed exposure to the inhibitor H7. Inhibitor was added at the times indicated by the red lines (left), and has progressively less effect to gene expression the later it was added to cells. RNase protection analysis for Apod, Eomes and Xbra expression. WE, whole embryos. (E) Quantitation of the gene expression over FGF-R ratio from the experiment shown in D.