Fig. 2. LB cells transfected with CD44v4-v10 cDNA express v4 and v6-encoded epitopes and bind soluble HA. (A) Parental LB cells (LB), LB cells transfected with CD44s cDNA (TRs) as well as LB cells transfected with CD44v4-v10 cDNA (TRv) and two representative clones (TRv2 and TRv3) of six derived from two separate transfections, were analyzed by flow cytometry for CD44 isoform expression, as described in Materials and Methods. The cells were stained with KM81 anti-pan CD44 mAb (marked CD44), 10D1 anti-CD44v4 mAb (marked V4) and 9A4 anti-CD44v6 mAb (marked V6) followed by anti-rat IgG conjugated to FITC. FITC indicates cells stained with the second antibody alone. Note that the pan CD44 fluorescence intensity of the TRs cells is higher than that of the other cell lines. (B) LB, TRs and TRv cells, as well as representative TRv cloned cells, were incubated with fluorescein-labeled HA (20 µg/ml) in the absence (Fl-HA) or presence ((Fl-HA+HA) of unlabeled HA (1 mg/ml), which competitively inhibits the binding of Fl-HA, and analyzed by flow cytometry. Segregated histograms display HA binding; matched histograms indicate the inability to bind HA. (C) All cells were incubated with fluorescein-labeled HA (20 µg/ml) in the absence (Fl-HA) or presence (Fl-HA+KM81) of KM81 (IgG2b) anti-CD44 mAb (100 µg/ml) and analyzed by flow cytometry. KM81 anti-CD44 mAb blocks the binding of Fl-HA to TRv cells. The histograms of the LB-TRo cells were almost identical to those of the LB cells (not shown).
