Western blot analysis was performed as described previously (Iacovelli et al. 2004). Briefly, EBs were either cultured for 8 days in the absence of any pharmacological treatment (controls) or subjected to a 4 –/4 + day treatment with RA or human recombinant Dkk-1. The reaction was stopped by washing twice with ice-cold phosphate-buffered saline and cells were lysed for 10 min at 4°C in Triton X-100 lysis buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10% glycerol, 1 mm phenylmethylsulphonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 10 mmβ-glycerophosphate). For the detection of β-catenin in cell nuclei, nuclear fractions were isolated by differential centrifugation using a commercially available kit (Pierce, Rockford, IL, USA). The purity of the fraction was verified using an antibody against the nuclear protein poly-ADP ribose polymerase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cell lysates were cleared by centrifugation (10 000 g for 10 min), and 40–80 µg protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted on to nitrocellulose and probed using different commercial antibodies. Membranes were saturated for 1 h with Tris-buffered saline (100 mm Tris, 0.9% NaCl) containing 0.05% Tween 20 and 5% non-fat dry milk, and then incubated overnight with primary antibodies. Antibodies were used at the following dilutions: rabbit Dlx-2 polyclonal antibodies (1 : 200; Chemicon); rabbit β-catenin polyclonal antibodies (1 : 1000; Cell Signaling Technology, Beverly, MA, USA,); mouse β-III tubulin monoclonal antibody (TuJ1, 1 : 1000; Covance, DBA, Milan, Italy), mouse β-actin monoclonal antibody (1 : 5000; Sigma Aldrich) and rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1 : 1000; Abcam, Cambridge, UK). Because EB differentiation was associated with changes in all housekeeping proteins we tested (β-actin, GAPDH), Dlx-2, β-III tubulin and β-catenin signals were normalized with respect to the amount of protein loaded in the gel stained with Pelican indian ink (1 µL/mL in 0.2% Tween-20 in Tris-buffered saline).