Notch family members expressed on different stages of ES cell differentiation. Whole cell lysates were prepared from ES cells or cells at different stages of differentiation. Western blot was performed using anti–Notch-1, -2, -3, and β-actin antibodies as described in “Materials and methods.” ES indicates embryonic stem cells cultured with LIF; day 7 and day 14, embryonic body development from ES cells for 14 days; day 20, ES cell DC generation for 6 days with GM-CSF and IL-3 after 14-day embryonic body development.