Increase in canonical Wnt signaling during neural differentiation. (a) Both TOP and Axin2 promoter luciferase activities in E14 ES cells were induced at 48 h after LIF removal. (b) Western blot analysis using ABC (active β-catenin) and β-actin antibodies. Active β-catenin level was the highest on day 4 of neural differentiation. (c) p-LRP level was the highest on day 4 of neural differentiation. (d)-(e) In Oct4-Gip/Ax2P-mCherry cells, only GFP expression was detected. mCherry expression was increased after 24 h addition of BIO (0.75 μM). (f)-(g) mCherry expression increased in the neural precursor region of TOP-mCherry ((d) and (e)) and Ax2p-mCherry ((f) and (g)) stable cell lines during neuronal differentiation. Stable cell lines were cultured in N2B27 medium for 14 days. Scale bars, 100 μm.