Presence of full-length and N-terminal trOPN in human BM. (A) Immunohistochemical analysis of human BM sections stained with an antibody specific for the “cryptic” binding site of N-terminal trOPN, demonstrated the restricted presence of N-terminal trOPN in the endosteal region. (B) Isotype control. (C) Identified thrombin, MMP-3, and MMP-7 cleavage sites in human OPN. (D) Western blot analyses using the antibody specific for the “cryptic” binding site of N-terminal trOPN. Data demonstrate recognition of human milk trOPN (lane tr, arrow) but no recognition of full-length human milk OPN (lane fl), or MMP-3 (lane 3), or MMP-7 (lane 7) cleaved human milk OPN. (E) Isotype control. (F) Western blot analysis demonstrating the presence of biotinylated fragments of human milk OPN present postcleavage with MMP-3 (lane 3) or MMP-7 (lane 7) detected using streptavidin. (G) Representative samples of bone lysates from 6 (P1 to P6) of 12 patients run under reduced conditions and immunoblotted with LF-124, an antibody that detects the N-terminal half of OPN. The N-terminal cleaved OPN (→, 30-32 kDa) is at least as dominant as the full-length (, approximately 55 kDa) form. (H) Neither of these 2 forms were detected in the isotype control.