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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.
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