Western blot
After preparation of ES cell extracts with M-Per (Pierce), cellular proteins were separated on sodium dodecyl sulfate (SDS)-14% polyacrylamide gels and transferred to nitrocellulose membranes (Millipore). Membranes were incubated with anti-ESG1 (1/500 dilution), anti-Oct3/4 (1/500; Santa Cruz Biotechnology), anti-CDK4 (1/200; Santa Cruz Biotechnology), and anti-GFP (1/1000; MBL) primary antibodies. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins (1/5000; Cell Signaling) were used to detect antibody binding. We visualized bound antibody with an ECL Western Blotting Detection System (Amersham).