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. 2009 Oct;90(5):463–479. doi: 10.1111/j.1365-2613.2009.00676.x

Figure 3.

Figure 3

Detection of aggrecan fragments released from IL-1α-stimulated cartilage by neoepitope antibodies. (a) The cleavage sites of aggrecan in the interglobular domain for MMPs and aggrecanases are shown. A further cleavage site for aggrecanases in the C-terminal region of aggrecan is also illustrated. (b) Porcine cartilage stimulated with control media or IL-1α at 0.1, 1, 10 and 100 ng/ml for 2 days is shown. The conditioned medium was harvested and analysed for GAG release using the DMMB assay (n = 3). (c, d and e) Equal volumes of conditioned media (50 μl) from the experiment shown in (b) were deglycosylated and Western blots performed for aggrecanase-cleaved neoepitopes using the BC-3 antibody (recognizing the N-terminal sequence 374ARGSV) [panel c], and the BC-14 antibody (recognizing the N-terminal sequence 342FFGVG) [panel d]. The Western blot on the far right in panel d shows purified aggrecan cleaved in vitro with recombinant MMP-3, deglycosylated and subjected to Western blotting using the BC-14 antibody.