Figure 2

Modified ES cells show proper expression of both wild-type and affinity-tagged alleles. (A and B) Expression of NTAP-Oct4, NSC-Oct4 and NFH-Oct4 detected by anti-Oct4 shows amount of Oct4 equivalent to that found in wild-type ES cells. (C) Expression of NSC-Oct4 detected by anti-S antibody. (D) Expression of NFH-Oct4 detected by anti-FLAG antibody. (E) Expression of NBH-Oct4 was detected by using streptavidin-horseradish peroxidase (HRP). Wild-type ES cells were loaded as negative controls. (F) Alkaline phosphatase (AP) staining shows that modified ES cells are pluripotent like control Oct4^WT/WT ES cells. (G) S-tag affinity purification was performed on Oct4^WT/NSC ES cells and Oct4^WT/WT ES cells. Western blot analysis was performed on the nuclear lysate (L) as well as on the unbound (U) fraction, the first wash (W₁), the last wash (W₄), the eluate (E) and the boiled beads (B) after elution of the purification process. The marker sizes are indicated in kilodaltons. Anti-β tubulin was used as a control.