Figure 4

Glucosamine inhibited N- glycosylation and auto- phosphorylation of EGFR in DU145 cells. (A) Western blot analysis of cells cultured in the presence of 2 mM glucosamine for 24 or 48 h. Whole-cell lysates were subjected to immunoblotting using antibodies specific for EGFR, phospho (Tyr845)-EGFR (pEGFR) and actin (loading control). The open arrow indicates the N-glycosylated receptor and its phosphorylated form, and the filled arrow indicates the N-glycosylation deficient receptor and its phosphorylated form. (B) Western blot analysis of the whole-cell lysates treated in vitro with peptide-N-glycosidase F (PNGase F). DU145 cells cultured with or without 2 mM glucosamine for 24 h, and then the whole-cell lysates were prepared and treated with or without peptide-N-glycosidase F (40 ug/ml) for 4 h at 37C followed by immunoblotting using antibodies specific for EGFR and actin (loading control). The open arrow indicates the molecular mass of N-glycosylated EGFR and the filled arrow indicates the reduced molecular mass of N-glycosylation deficient EGFR. Each blot is a representative of three independent experiments.