Fig. 5. B and B exert differential effects on the level of phosphorylated Smad2. (A) Embryos were injected at the one-cell stage with morpholinos (Mo) against B or B, or B mRNA as indicated. Embryos were harvested at stage 10, fixed, dissected through the lip and analysed by immunofluorescence using anti-Smad2 and anti-β-Catenin antibodies. The nuclei were visualised with DAPI. Parts a and b show an area from the ventral vegetal region and parts c-e show an area from the dorsal vegetal region. (B) Embryos remained uninjected (ui) or were injected with two doses of mouse B mRNA or mouse B mRNA, cultured until control embryos reached stage 9 and analysed by immunoblotting with anti-phospho-Smad2, Smad2/3 or phospho-ERK (pERK) antibodies. (C) Embryos were injected with distinct morpholinos (labelled 1 or 2) targeting B or B, respectively, or with a control morpholino, and analysed as in B. (D) Animal caps from stage 8 embryos were incubated with or without okadaic acid (OA, 25 nM) for 1 hour, treated with or without Activin for 20 minutes and processed for immunoblotting. (E) HeLa EGFP-Smad2 cells were transfected with either an siRNA SMARTpool control or a human B- or B-specific SMARTpool. Cells were incubated with TGF-β for the times indicated, fixed and visualised by confocal microscopy. (F) HeLa EGFP-Smad2 cells were transfected as in E and incubated with TGF-β for the times indicated. Samples were analysed by western blotting with anti-phospho-Smad2, anti-Smad2/3 and anti-pan B subunit antibodies.