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. 2020 Nov 5;10:19122. doi: 10.1038/s41598-020-75571-y

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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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Characterization of Gc1S, Gc2 and Gc1F synthesized by ExpiCHO-S cells in serum-free suspension culture. Gc1S and Gc2 were synthesized by ExpiCHO-S cells transfected with pcDNA3.4-TOPO^Gc1S-His or pcDNA3.4-TOPO^Gc2-His (Supplementary Fig. 1) in serum-free suspension culture. Gc1S and Gc2 were purified by His Trap HP (Ni²⁺ pre-charged) column chromatography and desalted by dialysis against 50 mM sodium phosphate pH 7.0 (desalted fraction). Sugar moieties of the desalted fraction alone (−) and after treatment with β-d-galactosidase and sialidase (+) were analyzed by blotting with biotin-conjugated HPA-lectin (arrowhead). Purified Gc1F was analyzed as a control. Gc1S, Gc2 and Gc1F reacted with anti-Gc antibody (lower column). Extra bands indicated by * were unknown HPA-lectin reactive proteins that became undetectable after vitamin D affinity column chromatography (see lanes 1F+, 1F−). (B) Gc1F, Gc1S and Gc2 synthesized by ExpiCHO-S cells in serum-free suspension culture were purified by His Trap HP (Ni²⁺ pre-charged) column chromatography and Vitamin D affinity column chromatography, and then desalted by dialysis against 50 mM sodium phosphate pH 7.0 (desalted fraction). Sugar moieties of the desalted fraction without treatment (−) and after treatment with β-d-galactosidase and sialidase (+) were analyzed by blotting with HPA, VVA, PNA, or MALII lectins as indicated. Full-length blots are included in a Supplementary Fig. 4.