Figure 3.

Detection of ConA and GNA binding to glycoproteins in S, R and T cells passaged three times in the absence or presence of either tunicamycin (0.1 μmol·dm⁻³) or GalNAc-α-O-benzyl (0.1 mmol·dm⁻³). Panel (a): Representative sample of flow cytometry measurement of FITC-ConA binding to S (upper plot), R (middle plot) and T (lower plot) cells. Black histogram – unlabeled S, R and T cells, red histogram – FITC-ConA-labeled S cells, blue histogram FITC-ConA-labeled R cells, and green histogram – FITC-ConA-labeled T cells. Parameter Δ (represents the difference between medians of labeled and unlabeled cells) was ascertained using this measurement. Similar measurements were obtained for S, R and T cells untreated and treated with tunicamycin and GalNAc-α-O-benzyl and documented in panels (b) (for ConA) and (c) (for GNA). Binding of lectin to untreated S cells was arbitrarily selected as 100%. White column—untreated cells; gray—column cell treated with GalNAc-α-O-benzyl and black column—cell treated with tunicamycin. Significance: * and +—value significantly differs from the corresponding value for S cells at p < 0.02 and p < 0.05, respectively. The data represent the means ± S.E.M. of five independent measurements. Panels (d) (for ConA) and (e) (for GNA) represent Eastern blot identification of glycoproteins in crude membrane fractions isolated from S, R and T cells untreated C, or treated with inhibitor of O-glycosylation (GalNAc-α-O-benzyl, O) or N-glycosylation (tunicamycin, N). Data are representative of three independent measurements. Red arrows indicate the P-gp form glycosylated with saccharides that are GNA ligands.