Figure 7.

Sulfation of HS chains is important for HS binding and endocytosis of 3D8 scFv. (a) Binding of 3D8 scFv and the Tat peptide to heparin and CMHs and (b) internalization of 3D8 scFv were analyzed in the presence of heparin and CMHs. (a) Competitive ELISA for heparin binding. A microtiter plate was coated with heparin (10 µg/ml) and binding of 3D8 scFv-pA or the Tat peptide to heparin in the presence of CMHs (20 μg/ml) was determined using rabbit IgG, followed by detection with AP-conjugated anti-rabbit IgG (a) and AP-conjugated streptavidin, respectively. Data represent the mean ± S.E. of triplicate wells and are representative of three independent experiments. (b) Confocal microscopy analysis of 3D8 scFv internalization. CHO-K1 cells were incubated) for 6 h at 37 °C with a 1:1 mixture of 3D8 scFv-pA (20 μg/ml) and CMHs (20 μg/ml). After fixation and permeabilization, cells were stained with rabbit IgG, followed by TRITC-conjugated goat anti-rabbit IgG. Nuclei were stained with Hoechst 33342 (blue). Bar, 5 μm. Data are expressed as the mean ± standard error of three independent experiments. All p values were calculated using a two-tailed Student’s t test. Statistical significance is indicated on the graphs (ns, p > 0.05; *p < 0.05; **p < 0.01).