Figure 1.

R10G-reactive sulfated glycans are present in the mouse pleural mesothelium. (A) Schematic representation of 6-sulfo di-LacNAc recognized by R-10G. C-6 sulfate (S), galactose (Gal), and N-acetylglucosamine (GlcNAc) are shown. The glycan is extended from variable underlying core glycans (R). (B) Sections of lungs from normal adult mice were co-stained with R-10G (red) and an anti-mesothelin antibody (upper, green) or an anti-laminin (lower, green), followed by Hoechst 33342 nuclear staining (blue). Shown are representative fluorescence microscopy images of the lower and central portions of the left lung lobe (n = 3). Dense R-10G staining signals in the pleural mesothelium (arrows) revealed by co-staining with the mesothelial marker mesothelin are presented. Plot profiles of R-10G and mesothelin staining or laminin staining are presented. Signal intensities along the line marker (white dashed line) paths in the merged images were determined. Scale bar: 20 µm. (C) GlcNAc-containing fractions of mouse lung lobes were obtained with wheat germ agglutinin-coated beads. The bead-bound materials were incubated without or with PNGase F. The immunoreactivity of R-10G was tested. Bands with molecular weights of >270 kDa were observed (closed arrowheads). E-cadherin was used to show protein equal loading and the successful pretreatment of PNGase F of the lung fraction. The 110 kDa band shifted from 120 kDa in the pretreated fraction (gray arrowhead). Bands with 240 kDa were also seen in IgG1 control blots (asterisk) [3].