Loss of Smad4 expression results in increased phosphorylation of R-Smads. A. Western blots of lysates from wild-type (CCE & CCB) and Smad4 null ES cells (FNN & BNN) were probed with antibodies directed against R-Smads Smad2/3 or phospho-Smad2. β-tubulin acts as a loading control. Steady-state levels of Smad2/3 are unperturbed but P-Smad2 expression is strongly upregulated in Smad4 null ES cells. TGF-β stimulated HepG2 cells were included as a positive control. B. Similarly P-Smad1/5/8 levels are markedly enhanced in Smad4 null ES cells, wheras steady state levels of Smad1 and Smad5 are unaffected. Bmp4 treated NIH3T3 cells were included as a positive control. As judged by unchanged P-Erk1/2 levels loss of Smad4 function has no detectable effect on Erk signalling.