Fig. 7. Heparan sulphate stimulates FN-independent binding of PGI/AMF at pH 5. (A) NIH-3T3 cells were left untreated or treated with 1, 10, 30 and 100 µg ml^–1 HS for 30 minutes at 37°C and then incubated for 30 minutes at 37°C with 25 µg ml^–1 PGI/AMF-568 in HEPES adjusted medium at pH 7.5 or 5 µg ml^–1 PGI/AMF-568 in MES adjusted medium at pH 5. Representative images show PGI/AMF-568 labeling in red and FN labeling in green in NIH-3T3 cells at pH 7.5 (B) or pH 5 (C) and PGI/AMF-568 labeling in FN^–/– cells (D) in control cells and cells treated with 10 µg ml^–1 HS. Bar, 20 µm. The bar graphs in B, C and D present the quantification of the fluorescent intensity of total cell-associated (Cell) and FN fibril-associated (FN) PGI/AMF-568 in the absence (CTL; white bars) or following pretreatment with 10 µg ml^–1 HS (+HS; green bars). In NIH-3T3 cells at pH 5 (C), PGI/AMF-568 binding was competed for by incubating the cells with 100 µg ml^–1 of unlabelled PGI/AMF before addition of 5 µg ml^–1 PGI/AMF-568 in complete medium adjusted to pH 5 in the absence (CTL comp; grey bars) or presence of 10 µg ml^–1 HS (HS comp; dark green). PGI/AMF-568 fluorescent intensity was quantified from 10 random images per condition and the data normalized to the condition presenting the maximum intensity (±s.e.m.; n=3; P<0.01).