FIGURE 7.
HS, OPG, and RANKL form a ternary complex. A, binding of purified RANKL (60 μg) to heparin-Sepharose. Dotted line represents the salt gradient. B, OPG dimer or purified OPG dimer-RANKL complex were bound to a heparin-Sepharose column and eluted with a salt gradient (dotted line). Fractions (16 to 19 ml, 0.5 ml/fraction) of the OPG-RANKL complex run were resolved by SDS-PAGE and visualized by silver staining. C, OPG monomer (20 μg), RANKL (20 μg), and H12 (2 μg) were incubated in different combinations as indicated for 4 h at 25 °C. The complexes were resolved on an SEC column. The elution positions of the Mr standards BSA, IgY, and ferritin are marked with black triangles (13.5, 12.05, and 11.05 ml, respectively). Two peak fractions of the ternary complex (11.8 ml peak) were visualized by silver stain. D, binding of RANKL or preformed RANKL-OPG complex (100 ng/ml) to MC3T3 cells determined by FACS. RANKL binding was detected by a phycoerythrin (PE)-conjugated anti-mouse RANKL mAb. E, binding of the RANKL-OPG complex to MC3T3 cells shown in double staining dot plot using anti-RANKL-PE and goat anti-mOPG followed by anti-goat IgG-Alexa 647.