Figure 4.

Amounts of AFP and its binding to 17β-estradiol in embryonic sera. (A) Gene expression analysis of AFP in WT and KO embryos at E16.5. The amplified copies of AFP were normalized against GAPDH, from which the ratio between copy numbers was determined to obtain normalized fold change. (B) Fetal mouse sera containing AFP (arrow) were visualized by 10% SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining, which were obtained from 25 each of wild-type and mutant embryos at E16.5. They were diluted 1/30 and loaded. (C) Embryonic serum proteins from the WT and the FucM deficient mice were subjected to 2-D electrophoresis, followed by CBB staining. After blotting onto a nitrocellulose membrane, lectin blotting was performed. Aleuria aurantia lectin (AAL) binds to N-linked oligosaccharides with L-fucose linked (α-1,3/α-1,6) to the proximal GlcNAc residue, while Ulex europaeus agglutinin I preferentially binds to Fucα(1-2)Galβ(1-4)GlcNAc. Spot intensities were analyzed using ImageJ to estimate relative fucosylation levels of AFP. (D) Scatchard analyses for the interactions of 17β-estradiol (E₂) with mouse embryonic sera are shown, which were determined by the dextran-coated charcoal (DCC) technique as described in the Methods section.