FIG. 4.

Rescue of stem cell renewal by EGFP fusion constructs. (A) Diagram of EGFP fusion constructs with wild-type and mutant gene products. The rescue index was determined as in Table 3. (B) Western blot analysis of nuclear extracts prepared from rescued transfectants with EGFPwt and EGFPΔN grown in the presence of Tc and unrescued transfectants with EGFPΔNΔC grown in the absence of Tc. Anti-Oct3/4N antibody detects wild-type Oct-3/4 in the latter but not the former, confirming the rescue events in EGFPwt and EGFPΔN transfectants. (C) Western blot analysis of the blot used in panel B with anti-GFP antibody. Proper production of EGFP fusion proteins was confirmed. (D) Photomicrographs of phase-contrast and fluorescent views of a stem cell colony rescued by the expression of EGFPwt (top) and of nucleus-localized EGFPΔNΔC in a stem cell colony grown in the absence of Tc (bottom). Nuclear fluorescence from EGFPwt is retained in differentiated cells produced after withdrawal of LIF (middle).