FIGURE 5.

Binding of monoclonal antibodies to slow migrating non-acid glycosphingolipids of human embryonic stem cells. A–D, thin-layer chromatogram after detection with anisaldehyde (A) and autoradiograms obtained by binding of the monoclonal anti-blood group A antibody HE-193 (B), the monoclonal anti-Le^x/SSEA-1 antibody P12 (C), and the monoclonal anti-Le^y antibody F3 (D). A–D, lane 1, slow migrating non-acid glycosphingolipids of human embryonic stem cell line SA181 (fraction 181:IV) (40 μg); lane 2, slow migrating non-acid glycosphingolipids of human embryonic stem cell line SA121 (fraction 121:IV) (40 μg); lane 3, reference A type 1 hexaglycosylceramide (GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer) (4 μg); lane 4, reference Le^x pentaglycosylceramide (Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer) (4 μg); lane 5, reference Le^y hexaglycosylceramide (Fucα2Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer) (0.2 μg); lane 6, reference Le^a pentaglycosylceramide (Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer) (4 μg); lane 7, reference Le^b hexaglycosylceramide (Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer) (4 μg); lane 8, reference H type 2 pentaglycosylceramide (Fucα2Galβ4GlcNAcβ3Galβ4Glcβ1Cer) (4 μg). The numbers to the left of the chromatogram in A denote the approximate number of carbohydrate residues in the bands. The chromatograms were eluted with chloroform/methanol/water (60:35:8, v/v/v), and the binding assays were done as described under “Experimental procedures”. Autoradiography was for 12 h.