J Clin Invest.
Namalwa-CD44v3-v10 and Namalwa-CD44vRA bind FGF-2 to a similar extent. (a) Flow cytometry. The indicated Namalwa transfectants were incubated with biotinylated FGF-2 in the absence (inset) or presence of 0.2 M NaCl and then analyzed by flow cytometry for their ability to bind this growth factor, detected by staining with streptavidin-PE. Control: Namalwa cells transfected with empty vector (Namalwa-neo) and incubated with biotinylated FGF-2. (b) Western blot analysis. Western blots of cell extracts from Namalwa transfectants with anti-FGF Ab confirmed the flow-cytometry analysis. The Namalwa transfectants were preincubated with FGF-2 before being subjected to cell extraction and gel electrophoresis. The anti-FGF Ab showed that FGF-2 was bound to a similar extent to CD44v3-v10 and CD44vRA, whereas CD44s did not bind FGF-2. Actin, a housekeeping gene product, is equally expressed in all transfectant extracts. (c) Excess soluble heparin blocks the binding of FGF-2 to Namalwa-CD44vRA. Namalwa-CD44vRA cells were coincubated with biotinylated FGF-2 and an excess of soluble heparin or soluble chondroitin sulfate A plus C, then analyzed by flow cytometry for their ability to bind the growth factor. The binding of biotinylated FGF-2 was detected with streptavidin-PE. The extreme left-hand histogram depicts Namalwa-CD44vRA cells incubated with streptavidin-PE only. Similar results were obtained using three Namalwa-CD44vRA clones (not shown). (d) Heparinase treatment reduces FGF-2 binding to Namalwa-CD44vRA. Namalwa-CD44vRA cells were treated with heparinase or chondroitinase ABC and then analyzed by flow cytometry for their ability to bind biotinylated FGF-2. Detection and control as in c. Similar results were observed in three Namalwa-CD44vRA clones (not shown).