Fig. 4. Erk2^-/- ES cells are severely deficient in neural and mesodermal commitment. (A) Immunoblotting for phospho-Erk1/2 (pErk1/2) and total Erk1/2 from serum-starved Erk2^+/- and Erk2^-/- mouse ES cells that were stimulated with foetal bovine serum (FBS), LIF, or unstimulated (-). (B-D) Immunostaining (left; phase-contrast, right) for nestin in Erk2^+/- cells (A), and two independent Erk2^-/- ES cell clones (B,C) fixed on day 6 of neural monolayer culture. Scale bar: 50 µm. (E) RT-PCR for Oct4, Fgf5, Rex1 and ß-actin (as loading control) on RNA isolated from two Erk2^-/- ES cell lines (B1, B3) that were maintained in N2B27 medium without LIF. Parallel cultures were exposed to LIF for 2 days (+LIF) before isolation of RNA. (F) RT-PCR analysis of wild-type and two Erk2^-/- (B1, B3) ES cell lines on day 4 of mesoderm monolayer differentiation. (G) FACS analysis for Pdgfr expression of wild-type and Erk2^-/- ES cells on day 5 of mesoderm monolayer differentiation.