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. 2012 Jun 21;287(34):29204–29212. doi: 10.1074/jbc.M112.371880

FIGURE 4.

FIGURE 4.

Transfer of GalNAc to GLuc-CTP constructs expressed in HEK 293T cells. A, HEK 293T cells were co-transfected with pCMV-GlucCG-CTP-CA(1–19)Myc-His or pCMV-GlucCG-CTP-CA(1–2)Myc-His and either control plasmid (lanes 1–3 and 7–9) or pcDNAI-C2GnT1 (lanes 4–6 and 10–12). The media were collected, and the products were digested with neuraminidase (Nase) or neuraminidase plus DP β-galactosidase as indicated. Following separation by SDS-PAGE and electrophoretic transfer to PVDF, the bands were visualized using anti-His. B, the amount of GalNAc transferred to the products of transfection of HEK 293T cells with pCMV-GlucCG-CTP-CA(1–19)Myc-His or pCMV-GlucCG-CTP-CA(1–2)Myc-His and either control plasmid or pcDNAI-C2GnT1 was compared by determining the amount of GLuc activity captured onto 96-well plates coated with WFA. The same samples were digested with neuraminidase to expose any GalNAc modified with α2,6-neuraminic acid. All assays were done in quadruplicate. Error bars indicate S.E. LU, light units.