Fig. 1. Analyses of heparan sulfate synthesis. (A) RT-PCR analyses of wild-type and Ext2^+/– trophoblast stem cells (TSC). RT-PCR using a forward primer for exon 2 and a reverse primer for exon 3 shows a band of the correct size in the control lanes (2 and 4). No band can be detected in the lane with DNA from Ext2^–/– TSC. When a forward primer against exon 8 and a reverse primer against exon 9 were used, a band in the lane with DNA from Ext2^–/– TSC (4) is visible. Amplification with primers for E-cadherin was used as a control. (B) Western blot analyses. Cell lysates from wild-type, Ext2^+/– and Ext2^–/– TSC were incubated with a polyclonal antibody against EXT2 and show expected bands of 82 kDa in cell lysates from wild-type and Ext2^+/– TSC. Lysates from Ext2^+/– TSC show a second band of lower molecular weight. The lysates from Ext2^–/– TSC show only the lower molecular weight protein. (C) ES cells derived from wild-type (left), Ext2^+/– (middle) and Ext2^–/– (right) embryos were grown in culture with ³⁵SO₄ and their GAGs were isolated. The blue symbols represent the recovery of counts before chondroitinase digestion, whereas the red points represent the recovery of counts after chondroitinase ABC digestion. Ext2^–/– cells did not produce HS. (D) 10E4, an antibody that recognizes HS was used on sections of E7.5 wild-type (left) and Ext2^–/– (middle) embryos. There is strong staining in the basement membranes of the wild-type embryo (arrowheads). Staining for HS was greatly diminished in Ext2^–/– mutants, but strong staining can be seen in the maternal deciduae (arrows). The right-hand image shows incubation with IgG as a negative control. Scale bars: 100 µm.