Characterisation of phospho-specific antibodies.

<p>(A) Characterisation of anti-phospho-Akt (pAkt) and anti-phospho-Erk (pErk) antibodies, the PI3K/Akt inhibitor LY294002 and FGF inhibitor SU5402 were used to inhibit Akt and Erk phosphorylation in gastrula embryos, respectively. 1% DMSO was used to exclude any possible interference from the inhibitor solvent. Anti-Erk (Erk), anti-Akt (Akt) and anti-α-tubulin (α-tubulin) were used as loading controls. (B) Characterisation of anti-phospho-Smad1/5/8 (pSmad1) and anti-phospho-Smad2 (pSmad2) antibodies. The TGF-βRI inhibitor SB505124 was used to inhibit Smad2 phosphorylation in gastrula embryos; injection of <i>wnt8a</i> mRNA was used to inhibit <i>bmp4</i> expression, thus preventing Smad1/5/8 phosphorylation. All inhibitors have been added at stage 6. 1% DMSO was used to exclude any possible interference from the inhibitor solvent. Smad2 and α-tubulin serves as internal controls to ensure equal loading in all lanes.