Figure 1.

CTB cross-links to endogenous gangliosides in lung but not colonic epithelial cells. (a) Cells cultured with Ac₄ManNDAz produce SiaDAz, a photocross-linking sugar that can be incorporated into glycoproteins and glycolipids in place of naturally occurring sialic acid. Cross-linking to fucosylated glycoproteins and to gangliosides can be assessed by evaluating the mobility of cross-linked CTB complexes by immunoblot, and by evaluating the sensitivity of cross-linking to inhibition by 2F-Fuc (for fucosylated glycoproteins) and NB-DGJ (for gangliosides). (b) T84 cells were cultured with Ac₄ManNDAz or ethanol in the presence or absence of 200 µM 2F-Fuc or 40 µg ml⁻¹ NB-DGJ for 3 days. Cells were then incubated with CTB, and UV irradiated. Cell lysates were analysed by 20% SDS–PAGE followed by immunoblot with anti-CTB antibody. β-Actin was used as a loading control. (c) The same as (b) but HBEC3 cells were used. Boxes highlight the effects of inhibitor (2F-Fuc or NB-DGJ) treatment on glycolipid–CTB cross-linking. (d,e) T84 and HBEC3 lysates from (b) and (c) were also analysed by 6% SDS–PAGE followed by immunoblot with anti-CTB antibody. Boxes highlight the effects of inhibitor (2F-Fuc or NB-DGJ) treatment on glycoprotein–CTB cross-linking. β-Actin was used as a loading control. Blots are representative images of two independent experiments. (Online version in colour.)