FIGURE 3.
5T-Fuc prevents the formation of sLeX on glycoproteins in HepG2 cells. A, the immunoblot analysis of HepG2 lysates prepared from cells exposed to increasing concentrations of 5T-Fuc demonstrated that 5T-Fuc inhibits sLeX expression on glycoproteins in a dose-dependent manner. Blots were probed with two sLeX-specific antibodies, first with CSLEX-1 and then, after stripping, with SNH3. Chinese hamster ovary (CHO) cell lysates were used as a negative control because these cells do not express the FUTs necessary to produce sLeX. B, PNGaseF-treatment (F) of HepG2 cell lysates indicates that the CSLEX-1 and SNH3 antibodies are reactive against sLeX contained within N-glycans. C, the relative concentration of GDP-Fuc decreased as cells were exposed to increasing concentrations of 5T-Fuc. CE peak areas (n = 3) are reported as mean ± S.E. *, p < 0.05; **, p < 0.0005. D, 5T-Fuc also treatment also decreased sLeX levels on glycoproteins secreted by HepG2 cells although glycoprotein fucosylation did not appear to be greatly affected when blots were probed with AAL. E, when cell lysates were treated with PNGaseF (F), those derived from cells exposed to 50 μm 5T-Fuc exhibited a reduced ability to bind to AAL.