Natural LOX-PP contains both O- and N- linked sugars. Pre-confluent MC3T3-E1 osteoblasts were treated with or without 400 pM TGF-β1 for 18 hours. Media proteins were collected, concentrated and deglycosylated (Materials and Methods). Deglycosylated samples were subjected to12% SDS PAGE gel followed by Western blotting with anti-LOX-PP antibody (19) resulting in a bands corresponding to Pro-LOX (A), and LOX-PP (B). Lane 1: media proteins from no TGF-β1 controls; lanes 2-6: media proteins from cultures treated with TGFβ-1. Lane 2: TGFβ-1 treated media proteins, lane 3: media proteins treated with O-glycosidases only (neuraminidase and O-glycosidase), lane 4: media proteins treated with PNGase F, lane 5: media proteins treated with both PNGase F and O-glycosidases.