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Fig. 1. Differentiation of ES cells is arrested in the absence of Erk1/2
signalling. (A) Activation of Sox1-EGFP expression during
monolayer differentiation of 46C cells monitored daily by flow cytometry under
different conditions (means of three experiments in duplicate±s.e.m).
(B) Western blotting for activated signal transduction cascade
components during differentiation in N2B27 media (representative blots).
(C) Efficiency of differentiation (relative proportion of
Sox1-EGFP-expressing cells) at day 3 in the presence of the factors
shown (normalised to the no-treatment control for each of three experiments
performed in duplicate +s.e.m.). (D) 46C cells were differentiated for
24 hours under the conditions shown, then lysed. Western blotting for
C-terminal phosphorylated Smad1, Smad5 and Smad8 (top) shows a robust response
to Bmp4 but no appreciable Smad activation in N2B27 containing 3 µM
PD184352 (arrows). Arrowhead indicates a non-specific contaminating band. S214
(linker; bottom) phosphorylation of Smad1 appears to not be affected by Erk1/2
inhibition in these conditions. (E) Real-time PCR on cDNA from
PDK1-/- and wild-type ES cells as well as from PDK1-/-
and wild-type cells after 4 days of monolayer differentiation. Results are the
expression levels relative to wild type at day 4 +s.e.m. ES, embryonic stem
cell.
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