Ep1264y

Cells were lysed with Laemmli sample buffer containing 2.5% β-mercaptoethanol and heated at 95°C for 5  min. Samples were loaded to 4–15% polyacrylamide gels (Bio-Rad) for electrophoresis. Proteins were then transferred to a Poly Vinylidene DiFluoride (PVDF) membrane (Merck), which was blocked with 5% dry milk, Tris buffered saline, 0.2% Tween, and incubated with primary antibodies (overnight at 4°C) followed by secondary antibodies (1:10000) for 1  hr at room temperature. Proteins were detected by using Luminata Crescendo (Merck) and LAS600 (GE Healthcare). The following antibodies were used: anti-MMP14 rabbit monoclonal (1:1000, EP1264Y, Abcam), anti-alpha-catenin rabbit monoclonal (1:1000, EP1793Y, Abcam), anti-Vimentin mouse monoclonal (1:1000, 1A4, Sigma), anti-Fibronectin rabbit polyclonal (1:1000, Sigma), anti-integrin β1 mouse monoclonal (1:1000, P5D2, Abcam), anti-integrin β3 rabbit monoclonal (1:1000, ERP17507, Abcam), and anti-actin mouse monoclonal antibody (1:2000, AC-40, Sigma).

The following primary Abs were used in this study: Aggrecan (6-B-4) (Abcam, Cambridge, MA, USA), ARGXX (BC-3) (Abcam), phospho-JNK1/2 (Thermo Fisher Scientific, Waltham, MA, USA) β-actin (Cell Signaling Technology, Danvers, MA, USA), FLAG-M2 (MilliporeSigma, Burlington, MA, USA), acetylated α-tubulin (6-11B-1) (MilliporeSigma), early endosome antigen 1 (EEA-1; Abcam), LRP-1 β-light chain (Abcam), LRP-1 α-heavy chain (8G1) (Abcam), a disintegrin and metalloproteinase (ADAM)-17 (Abcam), MMP-13 (Santa Cruz Biotechnology, Dallas, TX, USA), MMP14 (anti-catalytic domain EP1264Y) (Abcam), mouse monoclonal anti–MT1-MMP hemopexin domain 222-1D8 (a gift from Yoshi Itoh, Kennedy Institute) generated as previously described (41 (link)), and arl13b (ProteinTech, Rosemont, IL, USA). Anti-AGEG was a kind gift from Hideaki Nagase (University of Oxford). Purified aggrecan from bovine cartilage came from MilliporeSigma. Recombinant human C-terminal His-tagged human receptor-associated protein (RAP) was produced in Escherichia coli using a pET3a-based expression vector and purified as described previously (13 (link)). The domain deletion mutant, ADAMTS-5 (TS5-3)-flag, is described in Gendron et al. (42 (link)), and recombinant MMP-13 is described in Yamamoto et al. (43 (link)). Recombinant IL-1β was purchased from PeproTech (London, United Kingdom).

Primary antibodies used in this study: Rabbit polyclonal anti‐human FBN1 (HPA021057, used in immunofluorescence staining of MG63 cells; Sigma‐Aldrich), mouse monoclonal anti‐FN (610078; BD), mouse monoclonal anti‐RECK (5B; Takahashi et al., 1998), mouse monoclonal anti‐integrin β₁ (CD29; 610467; BD), mouse monoclonal anti‐integrin α₂ (611016; BD), mouse monoclonal anti‐integrin α₅ (CD49e: 610633; BD), mouse monoclonal anti‐FBN (pan; MAB2641: clone689, used in immunoblot assay, recognizes both FBN1 and FBN2; Merck Millipore), rabbit polyclonal anti‐MMP14/MT1‐MMP (EP1264Y: ab51074; Abcam), rabbit polyclonal anti‐ADAMTS10 (Matsuzaki et al., 2018), mouse monoclonal anti‐α‐tubulin (DM1A; Merck), and polyclonal horseradish peroxidase (HRP)‐conjugated goat anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; [V‐18] HRP; sc‐20357 HRP; Santa Cruz Biotechnology). Secondary antibodies: Anti‐rabbit immunoglobulin G (IgG)‐HRP (ab6721; Abcam), anti‐mouse IgG‐HRP (A4416; Sigma‐Aldrich), anti‐mouse IgG‐CF488 (20018; Biotium), and anti‐rabbit IgG‐CF647 (20282; Biotium).