FIG. 6.

Mouse Cripto is modified with the monosaccharide form of O-linked fucose on Thr-72. 293T cells were transiently transfected with HA-tagged Cripto or the T72A mutant. Following radiolabeling with [³H]fucose, proteins were isolated by immunoprecipitation with anti-HA antibodies. (A) Proteins were treated with or without PNGase F to remove N-glycans and were analyzed by Western blotting and fluorography. The large shift in molecular weight (apparent in the Western blot) after PNGase F treatment is due to the removal of N-glycans; the remaining radiolabel (apparent in the fluorograph) corresponds to O-linked fucose, which is absent in the T72A mutant. WT, wild type. (B) Following PNGase F treatment, the radiolabeled Cripto protein was analyzed by β-elimination and gel filtration chromatography to demonstrate that the O-linked sugar corresponds to the monosaccharide form of O-linked fucose (fucitol). The migration position of the tetrasaccharide form of O-linked fucose is indicated by TS.