Figure 2.

Glucosamine inhibited co-translational N-glycosylation of gp130 and glucose transporter activity was essential for the inhibition. (A) 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 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 gp130 and actin (loading control). The open arrow indicates the molecular mass of N-glycosylated gp130 without glucosamine or PNGase F treatment and the filled arrow indicates reduced molecular mass of N-glycosylation deficient gp130. (B) Western blot analysis of cells treated with 2 mM glucosamine in the presence or absence of cycloheximide. DU145 cells cultured with or without 2 mM glucosamine for 4 h in the presence or absence of cycloheximide (100 μg/ml), and then the whole-cell extracts were prepared and subjected to immunoblotting using antibodies specific for gp130 and actin (loading control). The open arrow indicates the molecular mass of N-glycosylated gp130 and the filled arrow indicates the reduced molecular mass of N-glycosylation deficient gp130. (C) Western blot analysis of DU145 cells treated with glucosamine in the presence or absence of glucose transporter inhibitor cytochalasin B. Cells pre-incubated with 10 μM cytochalasin B for 30 min and then treated with 2 mM glucosamine for 4 h. The whole-cell extracts were prepared and subjected to immunoblotting using antibodies specific for gp130 and actin (loading control). The open arrow indicates the molecular mass of N-glycosylated gp130 and the filled arrow indicates the reduced molecular mass of N-glycosylation deficient gp130. Each blot is a representative of three independent experiments.