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. Author manuscript; available in PMC: 2017 Oct 25.
Published in final edited form as: Arthritis Rheumatol. 2017 Apr 28;69(6):1246–1256. doi: 10.1002/art.40080

Figure 2. Soluble LRP1 ectodomain (sLRP1) prevents endocytosis of ADAMTS-5 and MMP-13 without interfering their activities.

Figure 2

(A and B) Human normal chondrocytes (n=3) were cultured with DMEM containing 10 nM ADAMTS-5 (A) or 10 nM MMP-13 (B) in the presence of 0–10 nM sLRP1, or 500 nM RAP for 0–4 h. ADAMTS-5 and MMP-13 in the medium were detected by Western blotting using anti-FLAG M2 antibody. Upper panel, representative Western blotting. Lower panel, quantified data. (C) Bovine aggrecan (0.5 mg/ml) was incubated with 0.05 nM ADAMTS-5 in the presence of 0–0.25 nM sLRP1 for 0–4 h at 37 °C. The reactions were stopped by 10 mM EDTA and the reaction products were deglycosylated and subjected to Western blotting using anti-ARGS aggrecan neoepitope antibody. Upper panel, representative Western blotting. Lower panel, quantified data. (D) Collagen II (1 mg/ml) was incubated with 5 nM MMP-13 in the presence of 0–25 nM sLRP1 for 0–3 h at 25 °C. The reactions were stopped by 10 mM EDTA and the reaction products were analyzed by SDS-PAGE staining with Coomassie Brilliant Blue. Upper panel, representative SDS-PAGE. Lower panel, quantified data. Data were expressed as the mean ± SD. *, p < 0.05, ****, p < 0.0001, by 2-tailed Student’s t test (A and B) or one-way ANOVA followed by Dunnett’s multiple comparison test (C).