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. 2026 May 6;11(19):28128–28140. doi: 10.1021/acsomega.5c12731

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© 2026 The Authors. Published by American Chemical Society

This article is licensed under CC-BY-NC-ND 4.0

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Neu5Gc is incorporated into polySia chains by Cma ST8Sia IV produced in HEK293 and HEK293S cells. Polysialylation reactions were performed in a filter unit overnight with or without 40 μM CMP-Neu5Gc. Polysialylated proteins were either (A) separated on SDS-PAGE, and WB was obtained with the mAb735 showing polysialylation obtained with ST8Sia IV produced in HEK293 (upper) and HEK293S cells (lower), or (B) treated (red boxplots) with endoN, collected in the flow-throughs, and analyzed by HPLC. As an additional control setup, the flow-throughs of samples without endoN were analyzed (blue boxplots). Samples from 3 independent experiments were measured (n = 3). The asterisks indicate the p-value: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; n.s. stands for not significant.