Fig. 1
Ucma physically interacts with ADAMTS5 and inhibits its aggrecanase activity in chondrocyte cultures. a Ucma–ADAMTS5 interactions investigated by slot blot binding assays: indicated amounts of recombinant ADAMTS5 blotted onto PVDF membrane incubated with recombinant FLAG-tagged Ucma (upper panel) or BSA (lower panel) and bound Ucma detected with anti-FLAG antibody and anti-mouse IgG-HRP. Collagen I and II blotted as negative and positive controls, respectively. b Solid phase assay to detect Ucma–ADAMTS5 interactions. ADAMTS5 and type I and type II collagen coated to multi-well ELISA plates at indicated doses and then incubated with Ucma. Detection of bound Ucma performed with anti-FLAG antibody and colorimetric detection system. Ucma protein interactions represented as oD at 492 nm corrected by oD492 of BSA-coated wells. c, d Chondrogenic 4C6 cell cultures treated with or without Ucma (doses indicated) and recombinant ADAMTS (20 nM) or IL-1β (2.5 ng/ml) for 24 h in serum-free medium and stained for ADAMTS-specific aggrecan cleavage product (NITEGE). NITEGE-positive chondrocyte culture area quantified using ImageJ software (d). Means ± SEM shown; n = 3 or 4. Representative data from three independent experiments each. Coll collagen, ADAMTS A disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats, BSA bovine serum albumin, Ctrl. control, IL interleukin, n/a not available, NITEGE ADAMTS-specific aggrecan neoepitope, Ucma Upper zone of growth plate and cartilage matrix-associated protein