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. 2020 Jun 15;37(4):485–498. doi: 10.1007/s10719-020-09931-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Western blot analysis of protein extracts from animal heart valves and pericardia using chicken IgY anti-Neu5Gc, and MAL-2 and SNA lectins (a). Recombinant mucin-type fusion proteins – CP-Gc carrying Neu5Gc structures, CP-ext C1 carrying extended core 1 O-glycans terminated with α2,3-linked sialic acid and CP-ext C1-ST6 carrying extended core 1 O-glycans terminated with α2,6-linked sialic acid – were used as positive controls for anti-Neu5Gc, MAL-2 and SNA staining, respectively. The C-P55 fusion protein lacking Neu5Gc was used as negative control for Neu5Gc staining. A human aortic endothelial cell lysate was used as negative control in all panels. Relative amounts of individual structures are given in percentage (%) of the total sum of integrated peak areas in the LC-MS chromatograms. Relative amounts of Neu5Gc-containing N- and O-glycans in the animal tissue samples are shown in (b). MS/MS spectra of two sulfated Neu5Gc-containing O-glycans with masses corresponding to a Neu5Gc₁Hex₁HexNAc₁Sul₁ ([M-H]^- of m/z 771) and a Neu5Gc₁Hex₂HexNAc₂Sul₁ ([M-H]^- of m/z 1136) structure are shown in c and d, respectively