Fig. 4. Involvement of Activin/Nodal, PI3-kinase and MAPK signaling pathways in β-catenin-induced mesoderm and the anterior PS differentiation. (A,C) Immunoblot analysis of total cell lysates from Nβ-cateninER cells. Cells were cultured for 3 days with or without 4OHT, Noggin (Nog; 250 ng/ml), Activin A (25 ng/ml) or BMP4 (10 ng/ml). Cell lysates were subjected to SDS-PAGE, and probed with specific antibodies as indicated. (B) The Nβ-cateninER cells were cultured with 4OHT and Noggin in the presence or absence of SB431542 (SB) for 3 days, and immunostained with anti-N-cadherin and FOXA2 antibodies. Scale bar: 100 µm. (D) Localization of β-catenin in Nβ-cateninER cells. Cells were cultured with or without 4OHT and Noggin, and subjected to immunostaining with anti-β-catenin (total) and anti-ER antibodies (to recognize exogenous Nβ-cateninER). Nuclei were counterstained with DAPI. Scale bar: 50 µm.