FIGURE 8.

Wnt receptor FZD-7 is N-glycosylated with β(1,6) branching. A, after human pCMV6-FZD7 plasmid was transiently expressed in LS180 cells, tagged FZD-7 proteins were detected by western-blotting (WB) using anti-DDK tag antibody. Note: denaturing samples by boiling at 95 °C caused aggregation of proteins, resulting in poor resolution. β-Actin was used as a loading control. B, LS180 cell lysates expressing FZD-7 were digested either with PNGase F or with Endo-H followed by SDS-PAGE and Western blotting using anti-DDK antibody. The arrowheads indicate a deglycosylated band of FZD-7 by PNGase F digestion. C, FZD-7 transfected LS180 cell lysates were used for precipitation with biotinylated L-PHA followed by the addition of streptavidin-agarose and elution by boiling in SDS-buffer followed by SDS-PAGE and Western blotting with anti-DDK tag antibody. D, after human pCMV6-FZD-7 plasmid was transiently expressed in Lec4 cells, tagged FZD-7 proteins were detected by Western blotting using anti-DDK tag antibody (left); after mouse GnT-V cDNA along with FZD-7 cDNA were transiently expressed in Lec4 cells, levels of N-linked β(1,6) branching were detected by L-PHA staining (middle panel); FZD-7 and GnT-V co-transfected Lec4 cell lysates were used for precipitation with biotinylated L-PHA followed by Western blotting with anti-DDK tag antibody (right panel).