3.

Cma ST8Sia IV produced in HEK293 and HEK293S cells can use CMP-Neu5Ac and CMP-Neu5Gc to elongate polySia chains. The precipitates of polysialylation reactions conducted with Cma ST8Sia IV produced in HEK293 cells or HEK293S cells with (A) 40 μM CMP-Neu5Ac (purple line) or (B) 40 μM CMP-Neu5Gc (blue line) were used for “mild” DMB labeling and analysis by anion exchange chromatography. In addition, a polysialylation reaction without an added donor substrate (black line) was performed. The chain lengths are given for selected peaks. Magnifications for visualizing chains in the DP 40 range are shown in Supplementary Figure S4.