Figure 10.

Removal of heparan sulphates only partly inhibits wtS1S2 binding. (A) RT4 cells were pretreated for 3 h with heparinase I/III mixture or control growth medium and then harvested by either trypsin/EDTA (+) or non-enzymatic cell dissociation solution (−) and then incubated with 100 nM wtS1S2 at 37 °C before staining with anti-His6 secondary antibody labelled with Dylight 488. The data are shown relative to the non-trypsinised- and non-heparinase-treated control and are the means ± SEM from 3–4 independent experiments performed in duplicate. Significance of difference from the control was assessed by a one-sample t test. ns = not significant; ** p < 0.001. (B) The effects of heparinase treatment was measured using antibody 3G10 that recognises the cleaved stubs of heparan sulphates. RT4 cells were pre-treated for 3 h with heparinase I/III mixture or control growth medium and then harvested by either trypsin/EDTA or non-enzymatic cell dissociation solution and then incubated with 3G10. Bound antibody was detected by an FITC-labelled secondary and cell-associated fluorescence was measured by flow cytometry. Data are the means ± SD from 2 separate experiments performed in duplicate.