Fig. 1. The MEKK1-MKK4 pathway leads to JNK and c-Jun phosphorylation in the developing eyelid epithelium. (A) Immunofluorescence staining of wild-type (WT), Mekk1^+/KD (M1^+/KD) and Mekk1^KD/KD (M1^KD/KD) fetuses at embryonic day 15.5 (E15.5) for p-MKK4 (top panels) and p-MKK7 (bottom panels) (green). Nuclei are stained with DAPI (blue). Scale bars: 50 µm. The images are representative of at least three fetuses of each genotype examined. (B) Mouse embryonic stem (ES) cells were either left untreated or treated with activin B (5 ng/ml) for 10 minutes or infected with adenovirus for WT-MEKK1 for 24 hours, as labeled. The cell lysates were subjected to western blot analyses using antibodies for phosphorylated and total MKK4 and MKK7, and MEKK1, as indicated. The results were quantified by chemiluminescence imaging and the fold induction of p-MKK4 and p-MKK7 by activin B and MEKK1 over the control was calculated. (C) Wild-type and Mkk4^-/- ES cells were infected with either control or WT-MEKK1 adenoviruses. At 24 hours post infection, cell lysates were examined by western blotting for phosphorylated and total JNK, MEKK1 and MKK4. (D) Immunofluorescence staining of wild-type (WT), Mekk1^+/KD (M1^+/KD) and Mekk1^KD/KD (M1^KD/KD) fetuses at embryonic day 15.5 (E15.5) detected phospho-JNK (p-JNK) (left panels) and phospho-c-Jun (p-c-Jun) (middle panels) (green) in the developing eyelid epithelium (arrowheads). Nuclei were stained with DAPI (blue). White rectangular areas of p-c-Jun staining (middle panels) are also shown at higher magnification (right panels). Dotted lines mark dermis (De), basal epithelial layer (Ep. B), suprabasal epithelial layer (Ep. S) and cornea (Co). Scale bars, 50 µm. At least four fetuses (8 eyes) of each genotype were examined. (E) The fraction of phosphorylation-positive cells over total cell count in suprabasal epithelial layer was calculated and statistically significant differences (P<0.05) from wild-type fetuses are denoted with an asterisk.