FIG. 1.

Neural differentiation and induction of NFLC protein in ES cells. Undifferentiated TC1 ES cells were submitted to the neural differentiation protocol described in Materials and Methods. (A) Cultures following 6 days of culture on polyornithine-laminin-coated dishes were photographed with a phase-contrast microscope revealing dense outgrowth of neurites from the attached EB in the lower right corner. (B) TC1 ES cell-derived neural cultures on polyornithine-laminin-coated slides were fixed and stained for NFLC as described in Materials and Methods. Staining was visualized with a fluorescence microscope at 400× magnification. (C) TC1 ES cells as well as wtES.1 ES cells derived in our lab were cultured as 2−/2+ EBs and then further cultured for 3 or 9 days on polyornithine-laminin-coated dishes. Whole-cell extracts from the indicated ES cells, 2−/2+ EBs, and neural cultures were prepared and immunoblotted for NFLC with the NR4 monoclonal antibody (Sigma Chemical Co.).