Fig. 4: ST6GAL1 K.O. triggers the competitive modification of ErbB2 N-glycan antennae with multi-fucosylated species.

A Sambucus nigra agglutinin (SNA) and Ulex europaeus agglutinin I (UEA-I) lectin blot analysis of ErbB2 carrying α2,6-linked sialic acid (α2,6NeuAc) and α1,2-linked fucose (α1,2Fuc), respectively, following receptor immunoprecipitation from NCI-N87 WT and ST6GAL1 K.O. whole cell lysates; B Colloidal Blue gel staining of immunoprecipitated WT and ST6GAL1 K.O. ErbB2. In all samples, the indicated band corresponding to ErbB2 was excised and further processed for glycomic analysis; C Illustrative example of peak quantification and isotopic distribution of an α2,6NeuAc-containing N-glycan species in the electropherograms of WT and ST6GAL1 K.O. ErbB2. Following receptor immunoprecipitation, in-gel Peptide N-Glycosidase F (PNGase F) digestion of the excised bands was performed to achieve total N-glycan release, followed by sialic acid derivatization, N-glycan purification and labeling with the Girard’s reagent P (GirP), and analysis by capillary electrophoresis–electrospray ionization-mass spectrometry (CE–ESI-MS); D Relative quantification of the released N-glycan species from WT and ST6GAL1 K.O. ErbB2; C1 clone 1, C2 clone 2, C3 clone 3.