Keratanase

Aggrecan digestion assay was performed as previously described [8 (link)]. Briefly, 50 μg of aggrecan (final concentration 670 nM) were incubated with ADAMTS5-2 (2 pM for cleavage at E¹⁷⁹⁰–A¹⁷⁹¹ site, 0.5 nM for cleavage at E³⁹²–A³⁹³ site) in TNC buffer at 37°C for 2 h. The reaction was stopped with EDTA buffer (200 mM sodium acetate, 250 mM Tris/HCl pH 8.0 and 100 mM EDTA). Aggrecan was incubated with 0.1 milliunits/μl of chondroitinase ABC and 0.1 milliunits/μl of keratanase (Seikagaku) overnight at 37°C to remove GAG chains. The samples were precipitated with cold acetone, incubated at–20°C for 4 h and then centrifuged at 13000 g for 30 min. The dried pellet was dissolved in reducing sample buffer, run on SDS/PAGE (6% gel) and analysed by Western blotting using Trans-Blot® TurboTM Transfer System (BioRad) according to the manufacturer's instructions. Membranes were probed with rabbit polyclonal anti-AGEG antibody (which detects aggrecanase cleavage at the E¹⁷⁹⁰–A¹⁷⁹¹ bond) [18 (link)] or mouse monoclonal BC-3 antibody (which detects aggrecanase cleavage at E³⁹²–A³⁹³ bond, Abcam).