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.