Fig. 2.

Decreased gene expression of Chst14 and HPLC profiles of DS and CS moieties in CS/DS chains of Chst14^−/− mice. (A) Relative mRNA levels of Chst14 and Chst11 in placentas of wild-type (+/+), heterozygous (+/−) and homozygous (−/−) fetuses (means + SEM, n = 6/group). ***P < 0.001, compared with wild-type; ^###P < 0.001, compared with heterozygous; one-way ANOVA followed by the Tukey–Kramer post hoc test. (B–G) HPLC profiles of the digests of DS (B–D) and CS (E–G) moieties in CS/DS chains prepared from placentas of wild-type (B, E), heterozygous (C, F) and homozygous (D, G) fetuses following digestion with chondroitinase B (B–D) or AC (E–G) into disaccharides for analysis of DS or CS moieties, respectively. Each digest was labeled with 2AB, and 2AB-labeled CS/DS disaccharides were separated by anion-exchange HPLC on an amine-bound silica PA-G column using a linear gradient of NaH₂PO₄, as indicated by the dashed line. The amount of resultant disaccharides in each sample was calculated based on the peak area in reference to that of standard unsaturated disaccharides. The elution positions of authentic 2AB-labeled CS/DS disaccharides are indicated by the numbered arrows: 1, ΔHexUA-GalNAc; 2, ΔHexUA-GalNAc(6S); 3, ΔHexUA-GalNAc(4S); 4, ΔHexUA(2S)-GalNAc(6S); 5, ΔHexUA-GalNAc(4S,6S); 6, ΔHexUA(2S)-GalNAc(4S,6S). *Peak of ΔHexUA-GlcNAc (degradation product of hyaluronic acid). Peaks detected between standards 1 and 2 (indicated by #) are impurities that were also detected in chromatograms of the negative controls without enzymatic digestion (data not shown).