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. 2016 Jan 22;291(11):5708–5720. doi: 10.1074/jbc.M115.712836

FIGURE 1.

FIGURE 1.

Overexpression of GnT-III in MDA-MB-231 cells significantly inhibited the α2,3-sialylation, but not α2,6-sialylation, at a post-transcriptional level. A, cell lysates from control and GnT-III transfected MDA-MB-231 cells were immunoblotted with E4-PHA and DSA lectins or immunoprecipitated (IP) with ConA-agarose and blotted with ConA, MAA, and SNA lectins. B, to confirm the effect of GnT-III expression on cell sialylation, cells transfected with or without GnT-III were incubated with (bold line) or without (gray shading) biotin-conjugated MAA (recognizing α2,3-sialylated proteins), followed by incubation with streptavidin Alexa Fluor 647 conjugate and subjected to FACS analysis. C, to determine the changes in the N-glycans on specific proteins after GnT-III overexpression, the cell lysates from control and GnT-III transfected MDA-MB-231 cells were also immunoprecipitated by ConA, E4-PHA, MAM (recognizing α2,3-sialylated proteins), and S. sieboldiana (recognizing α2,6-sialylated proteins) agaroses and probed with antibodies against α3 integrin, αv integrin, and β1 integrin separately. D, PA oligosaccharides treated with or without glycosidases from those cells were analyzed by reversed phase HPLC. E, PA oligosaccharides treated with or without neuraminidases from the control, GnT-III transfected MDA-MB-231 cells, as well as their ST6GAL1 knock-out counterparts, were analyzed by anion exchange HPLC to quantify the amount of sialylated N-glycans. F, RT-PCR using total RNA extracted from the control and GnT-III transfected MDA-MB-231 cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. SCT, sialic acid transporter; St3gal, β-galactoside α2,3-sialyltransferase; St6galnac, α-N-acetylgalactosaminide α2,6-sialyltransferase; NEU, neuraminidase. Con, control (DOX-inducible GnT-III-overexpressing cells without DOX treatment); OE, DOX inducible GnT-III-overexpressing cells treated with DOX; ST6GAL1 KO, ST6GAL1 knock-out cells.