Fig. 5. hLTBP-3 is secreted by osteosarcoma cells as high molecular weight complexes. (A) Concentrated cell culture medium of MG-63, U-2OS and Saos-2 osteosarcoma cells was collected after culturing for 3 days under serum-free conditions. The protein samples were separated by 7.5% SDS-PAGE under non-reducing conditions and blotted onto nitrocellulose filters. Conditioned mediums from untransfected COS-7 cells and COS-7 cells overexpressing hLTBP-3 and TGF-ß1 were used as controls. Immunodetection was carried out using the affinity-purified anti-hLTBP-3 (Ab-hL3/2925), (B) anti-ß1•LAP antibody (680) or (C) anti-LTBP-1 (ab39) antibodies. The signals for A and B are from the same filter, which was recycled. Black arrows indicate the large latent complexes of hLTBP-3 and ß1•LAP, whereas white arrows indicate the large latent complexes of LTBP-1 and ß1•LAP.