Figure 1

Changes in disialyl-T structure in each tissue. (A) Schematic representation of the putative biosynthetic pathway of disialyl-T antigen. Glycosyltransferases involved in biosynthesis are indicated. ppGalNAc-Ts: polypeptide N-acetylgalactosaminyltransferases, C1galt1: Core 1 β1,3-galactosyltransferase, St3gal1: β-galactoside α2,3-sialyltransferase 1, St6galnac: GalNAc α2,6-sialyltransferase. (B) Genomic structure of the St6galnac3 and St6galnac4 genes and the sequences (arrows) targeted by the guide RNAs. (C) Genotyping of the St6galnac3 and St6galnac4 genes. DNA extracted from the tail of each mouse was used as the template for PCR. WT: wild type, Hz: heterozygous, KO: knockout, M: DNA marker. (D) HE and MALII lectin (red) staining using major tissues of WT and DKO mice. The results of other tissues are shown in Fig. S2. (E) MALII lectin blot of major tissues from WT and DKO mice. Coomassie brilliant blue-stained acrylamide gels are shown as loading controls. W: WT, D: DKO, SM: Skeletal muscle, Duo: Duodenum, Ep: Epididymis. (F,G) Dot blot analysis of brain extracts using MALII (F) and Jacalin (G) lectin treated with α2–3,6,8,9 Neuraminidase A (sialidase) or buffer as control. Each sample was tested in triplicates (left panel). The intensities of dot blots were measured using ImageJ (right panel). W: WT, 3: 3KO, 4: 4KO, D: DKO. n = 3, Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001. (H) MALII lectin blotting using the spleen extracts of WT, 3KO, 4KO, and DKO mice. Arrowhead: Bands with a significantly reduced signal. W: WT, 3: 3KO, 4: 4KO, D: DKO.