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. 2024 Jun 4;300(7):107450. doi: 10.1016/j.jbc.2024.107450

Figure 6.

Figure 6

Effects of LDN on terminal modifications of N-glycans.A, flow cytometry analysis of HEK293 DKO cells expressing B4GALNT3 with MAM and SNA lectins. B4GALNT3-expressing cells without lectin staining were used as a negative control (Lectin [−]). B, GnGnbi-PA was incubated with purified B4GALT1 or vehicle with UDP-Gal (upper) or with purified B4GALNT3 or vehicle with UDP-GalNAc (lower). Then, ST3GALT4, ST6GAL1, or vehicle and CMP-NeuAc were added for the second step reaction. The reaction mixture was analyzed by reverse-phase HPLC. C, HeLa cells were transfected with the expression plasmid for FUT2-myc or FUT4-myc with or without the plasmid for B4GALNT3. The cell lysates were blotted with anti-B4GALNT3, anti-myc, anti-GAPDH, and AAL. The signal intensity of the bands (>20 kDa) blotted with AAL was quantified (right graph). D, HeLa cells were transfected with the expression plasmid for GlcAT-P and HNK-1ST with or without the plasmid for B4GALNT3. The cell lysates were blotted with anti-B4GALNT3, anti-GlcAT-P, anti-GAPDH, and HNK-1 mAb. The signal intensity of the bands (>20 kDa) blotted with HNK-1 mAb was quantified (right graph). AAL, Aleuria aurantia lectin; DKO, double KO; HEK293, human embryonic kidney 293 cell line; HNK, human natural killer; LDN, LacdiNAc; MAM, Maackia amurensis lectin; SNA, Sambucus nigra lectin.